Antibody producing non-human mammals

ABSTRACT

Described are transgenic, non-human animals comprising a nucleic acid encoding an immunoglobulin light chain, whereby the immunoglobulin light chain is human, human-like, or humanized. The nucleic acid is provided with a means that renders it resistant to DNA rearrangements and/or somatic hypermutations. In one embodiment, the nucleic acid comprises an expression cassette for the expression of a desired molecule in cells during a certain stage of development in cells developing into mature B cells. Further provided is methods for producing an immunoglobulin from the transgenic, non-human animal.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/589,181, filed Oct. 19, 2009, pending, which application is acontinuation of U.S. patent application Ser. No. 12/459,285, filed Jun.29, 2009, pending, which applications claim the benefit, under 35 U.S.C.§119(e), to U.S. Provisional Patent Application Ser. No. 61/133,274,filed Jun. 27, 2008, for “Antibody Producing Non-Human Mammals,” theentire contents of each of which are hereby incorporated herein by thisreference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)-SEQUENCE LISTINGSUBMITTED AS ASCII TEXT FILE

Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT versionand a PDF version of the Sequence Listing have been submittedconcomitant with this application, the contents of which are herebyincorporated by reference.

TECHNICAL FIELD

The invention relates to the production and use of non-human animalscapable of producing antibodies or derivatives thereof, which areexpressed from at least partially exogenous nucleic acids (transgenes).Transgenes to produce such transgenic animals and methods to producesuch heterologous antibodies; methods and vectors for producing suchtransgenic animals are disclosed.

BACKGROUND

B cells mediate humoral immunity by producing specific antibodies. Thebasic structural subunit of an antibody (Ab) is an immunoglobulin (Ig)molecule. Ig molecules consist of a complex of two identical heavy (H)and two identical light (L) polypeptide chains. At the amino terminus ofeach H chain and L chain is a region that varies in amino acid sequencenamed the variable (V) region. The remaining portion of the H and Lchains is relatively constant in amino acid sequence and is named theconstant (C) region. In an Ig molecule, the H and L chain V regions (VHand VL) are juxtaposed to form the potential antigen-binding site. Thegenes that encode H and L chain V regions are assembled somatically fromsegments of germline DNA during precursor B (pre-B) celldifferentiation: V, D and J gene segments for the H chain and V and Jgene segments for the L chain. Within Ig V regions are three regions ofgreatest amino acid sequence variability that interact to form theantigen-recognition site and are thus referred to as complementaritydetermining regions (CDRs).

The V gene segment encodes the bulk of the V region domain, includingCDR1 and CDR2. Diversity in CDR1 and CDR2 derives from sequenceheterogeneity among multiple different germline-encoded V segments. CDR3is encoded by sequences that are formed by the joining of H chain V, D,and J gene segments and L chain V and J segments and by mechanisms thatcreate nucleotide sequence heterogeneity where these segments arecombined. Additional diversity may be derived from pairing of differentH and L chain V regions. Collectively these processes yield a primaryrepertoire of antibodies encoded by germline gene segments and expressedby newly formed B cells.

An additional source of antibody diversity is imposed on top of thediversity generated by recombination of Ig gene segments. B cells areable to introduce mutations into the antibody V regions that theyexpress, a process called somatic hypermutation. Thus, when an animalfirst encounters an antigen, the antigen binds to a specific B cellwhich happens to carry antibodies which have a V domain which binds theantigen. This primary response may activate this B cell to go on tosecrete the cognate antibody. These activated B cells can also nowtarget a somatic mutation process to their rearranged antibody genesegments and thus allow the production of daughter cells which makevariants of the antibodies of the primary response. A selection processamplifies those variant B cell descendants which make an antibody ofimproved affinity of the antigen. In B cells, somatic hypermutations aretargeted to a restricted genomic region including both the rearranged VHand VL genes. Thus somatic mutation allows affinity maturation—theproduction and selection of high affinity antibodies. Therefore, somaticmutation is important for the generation of high affinity antibodies.

The exquisite specificity and high affinity of antibodies and thediscovery of hybridoma technology allowing the generation of monoclonalantibodies (mAbs) has generated great expectations for their utilizationas targeted therapeutics for human diseases. MAbs are identical becausethey are produced by a single B cell and its progeny. MAbs are made byfusing the spleen cells from a mouse that has been immunized with thedesired antigen with myeloma cells to generate immortalized hybridomas.One of the major impediments facing the development of in vivoapplications for mAbs in humans is the intrinsic immunogenicity ofnon-human Igs. Patients respond to therapeutic doses of mouse mAbs bymaking antibodies against the mouse Ig sequences (Human Anti MouseAntibodies; HAMA), causing acute toxicity, alter their biodistributionand accelerate clearance, thus reducing the efficacy of subsequentadministrations (Mirick et al. (2004), Q. Nucl. Med. Mol. Imaging48:251-257).

To circumvent the generation of HAMA, antibody humanization methods havebeen developed in an attempt to produce mAbs with decreasedimmunogenicity when applied to humans. These endeavors have yieldedvarious recombinant DNA-based approaches aimed at increasing the contentof human amino acid sequences in mAbs while retaining the specificityand affinity of the parental non-human antibody. Humanization began withthe construction of mouse-human chimeric mAbs (S. L. Morrison et al.(1984), Proc. Natl. Acad. Sci. USA 81:6851-5), in which the Ig C regionsin murine mAbs were replaced by human C regions. Chimeric mAbs contain60-70% of human amino acid sequences and are considerably lessimmunogenic than their murine counterparts when injected into humans,albeit that a human anti-chimeric antibody response was still observed(W. Y. Hwang et al. (2005), Methods 36:3-10).

In attempts to further humanize murine mAbs, CDR grafting was developed.In CDR grafting, murine antibodies are humanized by grafting their CDRsonto the VL and VH frameworks of human Ig molecules, while retainingthose murine framework residues deemed essential for specificity andaffinity (P. T. Jones et al. (1986), Nature 321:522). Overall,CDR-grafted antibodies consist of more than 80% human amino acidsequences (C. Queen et al. (1989), Proc. Natl. Acad. Sci. U.S.A.86:10029; P. Carter et al. (1992), Proc. Natl. Acad. Sci. U.S.A.89:4285). Despite these efforts, CDR-grafted, humanized antibodies wereshown to still evoke an antibody response against the grafted V region(W. Y. Hwang et al. (2005), Methods 36:3).

Subsequently to CDR grafting, humanization methods based on differentparadigms such as resurfacing (E. A. Padlan et al. (1991), Mol. Immunol.28:489), superhumanization (P. Tan D. A. et al. (2002), J. Immunol.169:1119), human string content optimization (G. A. Lazar et al. (2007),Mol. Immunol. 44:1986) and humaneering have been developed in an attemptto further decrease the content of non-human sequences in therapeuticmAbs (J. C. Almagro et al. (2008), Frontiers in Bioscience 13:1619). Asin CDR grafting approaches, these methods rely on analyses of theantibody structure and sequence comparison of the non-human and humanmAbs in order to evaluate the impact of the humanization process intoimmunogenicity of the final product. When comparing the immunogenicityof chimeric and humanized antibodies, humanization of variable regionsappears to decrease immunogenicity further (W. Y. Hwang et al. (2005),Methods 36:3-10).

De-immunization is another approach developed to reduce theimmunogenicity of chimeric or mouse antibodies. It involves theidentification of linear T-cell epitopes in the antibody of interest,using bioinformatics, and their subsequent replacement by site-directedmutagenesis to human or non-immunogenic sequences (WO 9852976 A1, thecontents of which are incorporated by this reference). Althoughde-immunized antibodies exhibited reduced immunogenicity in primates,compared with their chimeric counterparts, some loss of binding affinitywas observed (M. Jain et al. (2007), Trends in Biotechnol. 25:307).

The development of phage display technology complemented and extendedhumanization approaches in attempts to obtain less immunogenic mAbs fortherapy in humans. In phage display, large collections (“libraries”) ofhuman antibody VH and VL regions are expressed on the surface offilamentous bacteriophage particles. From these libraries, rare phagesare selected through binding interaction with antigen; soluble antibodyfragments are expressed from infected bacteria and the affinity ofbinding of selected antibodies is improved by mutation (G. Winter et al.(1994), Annu. Rev. Immunol. 12:433). The process mimics immuneselection, and antibodies with many different bindings specificitieshave been isolated using this approach (H. R. Hoogenboom et al. (2005),Nat. Biotechnol. 23:1105). Various sources of H and L chain V regionshave been used to construct phage display libraries including thoseisolated from non-immune or immune donors. In addition, phage displaylibraries have been constructed of V regions that contain artificiallyrandomized synthetic CDR regions in order to create additionaldiversity. Often, antibodies obtained from phage display libraries aresubjected to in vitro affinity maturation to obtain high affinityantibodies (H. R. Hoogenboom et al. (2005), Nat. Biotechnol. 23:1105).

The creation of transgenic mouse strains producing human antibodies inthe absence of mouse antibodies has provided another technology platformfor the generation of specific and high affinity human mAbs forapplication in humans. In these transgenic animals, the endogenous mouseantibody machinery is inactivated and replaced by human Ig loci tosubstantially reproduce the human humoral immune system in mice (A.Jakobovits et al. (2007), Nat. Biotechnol. 25:1134; N. Lonberg (2005),Nat. Biotechnol. 23:1117). B cell development as well as Igdiversification by recombination of gene segments is faithfullyreproduced in these mice, leading to a diverse repertoire of murine Bcells expressing human Igs. By immunizing these mice with antigens, itwas further demonstrated that these transgenic animals accumulatedsomatic mutations in the V regions of both heavy and light chains toproduce a wide diversity of high-affinity human mAbs (N. Lonberg (2005),Nat. Biotechnol. 23:1117).

The question, whether “fully human” mAbs such as derived from phagedisplay libraries or transgenic mice are less immunogenic than humanizedmAbs cannot be answered yet, because full immunogenicity data areavailable for just two human mAbs. An anti-tumor necrosis factor mAb,developed from phage-displayed human libraries induced antibodyresponses in 12% of patients—at the higher end of the incidence ofanti-antibody responses of the humanized antibodies (W. Y. Hwang et al.(2005), Methods 36:3-10).

Evaluation of the immunogenicity of the first registered human mAbgenerated by the transgenic approach demonstrated that mAb treatmentresulted in the generation of antibodies in approximately 5.5% oftreated cancer patients (A. Jakobovits et al. (2007), Nat. Biotechnol.25:1134; J. A. Lofgren et al. (2007), J. Immunol. 178:7467).

DISCLOSURE OF THE INVENTION

Disclosed are a method and means for producing antibodies that arespecific for their targets, but are less immunogenic. Described herein,the reduction of immunogenicity is at least partially achieved byproviding a transgenic non-human mammal comprising, at least in its Bcell lineage, a nucleic acid encoding at least an immunoglobulin lightchain or heavy chain, wherein the heavy- or light chain encodingsequence is provided with a means that renders it resistant to DNArearrangements and/or somatic hypermutations, preferably such anon-human animal is a rodent, more specifically a mouse. In certainembodiments, the nucleic acid encodes a human, human-like, or humanizedimmunoglobulin chain.

In the remainder of this specification, mice are typically used asexamples of the non-human mammals. The transgenic, non-human, mammalianhosts are capable of mounting an immune response to an antigen, wherethe response produces antibodies having primate, particularly human,variable regions. Various transgenic hosts may be employed, particularlymurine, lagomorpha, ovine, avine, porcine, equine, canine, feline, orthe like. Mice have been used for the production of B-lymphocytes forimmortalization for the production of antibodies. Since mice are easy tohandle, can be bred in large numbers, and are known to have an extensiveimmune repertoire, mice will usually be the animal of choice. Therefore,in the following discussion, the discussion will refer to mice, but itshould be understood that other animals, particularly non-primatemammals, may be readily substituted for the mice, following the sameprocedures.

The reason for preventing rearrangements and hypermutation is that inthis manner a non-immunogenic polypeptide can be chosen beforehandknowing that this polypeptide chain will remain non-immunogenic. Atleast one of the chains of the resulting immunoglobulin is thus lessimmunogenic. The resulting antibody needs to have (usually) both alight- and a heavy chain. The non-immunogenic chain must therefore becapable of pairing with the other chain. The other chain may be anendogenous chain, an exogenous chain or a hybrid of both. For humantherapy, the non-immunogenic chain should be as close to human aspossible.

A means for rendering a gene encoding an immunoglobulin chain (orchains) resistant to DNA rearrangement and/or mutation is of courseremoval of all genetic elements responsible for the rearrangement and/ormutation. The drawback thereof is that the variability of the two chainsis eliminated, whereas the invention preferably retains the variabilityin one chain (preferably the heavy chain) and inhibits and/or preventsthe rearrangement-mutation of the other chain (preferably the lightchain).

The elements for rearrangement and/or hypermutation characterized so farare located within the loci for immunoglobulins. Therefore the means forrendering the immunoglobulin encoding sequence resistant to DNArearrangement and/or mutation is inserting the gene in a locus outsidethe immunoglobulin loci.

Thus, described herein, a transgenic non-human mammal is providedwherein the light/heavy chain encoding sequence is integrated in thegenome of the non-human mammal in a locus outside the immunoglobulinloci. Preferably the insertion is in a locus that is resistant to genesilencing. Described herein, the integration is in the Rosa-locus or acomparable locus.

In certain embodiments, provided is an expression cassette that can beinserted into a Rosa locus or comparable locus with a means that allowsexpression of the immunoglobulin chain(s) essentially limited to cellsof B cell lineage, preferably with a means that allows expression of thelight chain encoding nucleic acid during a certain stage of thedevelopment of B cells. The term “essentially limited expression”indicates that expression is predominantly in cells of the B-celllineage, but that lower levels of expression in other cells, as comparedto the level of expression in B-cells, is possible. In certainembodiments, the term “essentially limited expression” indicates thatthe expression is exclusively present in cells of the B-cell lineage.Such means typically and preferably include B cell (developmental stage)specific promoters such as CD19, CD20, μHC (all V-genes), VpreB1,VpreB2, VpreB3, λ5, Igα, Igβ, κLC (all genes), λLC (all genes), BSAP(Pax5). Although it is very well possible to direct the expression ofthe DNA rearrangement and/or mutation resistant chain by such promoters,they are relatively weak. A strong promoter will typically be requiredto ensure adequate surface expression of the B cell receptor (made up ofthe membrane attached Ig H and L chain) and to compete with theexpression and pairing of endogenous chains (if present) through allelicexclusion. Such a promoter, however is usually not tissue specific. Toconfer tissue specificity, an indirect system employing Cre/lox or thelike is preferred. The desired chain is put under control of a strongpromoter inhibited by an element that can be removed by the action of aCre-protein, leading to activation of the desired immunoglobulinencoding gene. This system is described in detail in F. T. Wunderlich(2004), “Generation of inducible Cre systems for conditional geneinactivation in mice,” Inauguraldissertation zur Erlangung desDoktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät derUniversitat zu Köln; on the internet atdeposit.ddb.de/cgi-bin/dokserv?idn=97557230x&dok_var=d1&dok_ext=pdf&filename=97557230x.pdf.

Preferably the immunoglobulin chain produced in a manner resistant torearrangements and hypermutation is a light chain capable of pairingwith different heavy chains encoded by the non-human mammal. The lightchain will then be the same (and less immunogenic) in all antibodies,but variety in specificity is retained through rearrangements andhypermutations in the heavy chains. It may in that case be preferable tosilence at least one of the endogenous loci encoding a light chain,although allelic exclusion may render this unnecessary.

According to this embodiment, preferably the endogenous kappa (κ) lightchain locus is functionally silenced.

If the endogenous κ light chain locus is silenced, but also for otherreasons, it is preferred that the resistant light chain is a κ lightchain, preferably a light chain that has a germline-like sequence.Described herein such a light chain would lead to an antibody withreduced immunogenicity. The preferred germline sequence is based on thehuman IGKV1-39 (O12) as this light chain is very frequently observed inthe human repertoire (de Wildt et al. 1999, J. Mol. Biol. 285(3):895)and has superior thermodynamic stability, yield and solubility (Ewert etal. 2003, J. Mol. Biol. 325(3):531).

The following gives more specific embodiments of the expression cassettewith which the non-human animal can be provided described herein.Although this is typically advantageous for immunoglobulins, other genesof interest are also contemplated.

Thus, provided in a specific embodiment, is a transgenic non-humanmammal wherein the light chain encoding nucleic acid comprises in 5′-3′direction: a B cell specific promoter, a leader, a rearranged human Vgene, optionally a mouse κ-intron enhancer (MoEκi), a constant region(κ) and optionally a (truncated) mouse κ-3′ enhancer (MoEκ3′). Neubergeridentified and examined a novel B-cell specific enhancer locateddownstream of the kappa constant region (Neuberger, EP 00469025 B1, thecontents of which are incorporated herein by this reference). Thisenhancer has been shown to play a crucial role in the expression ofkappa genes as removal of the 808 bp enhancer strongly reducedexpression. Deletion of the 3′ kappa enhancer also strongly reduced thelevel of somatic hypermutations (SHM). In transgenic and cell expressionstudies, it has been revealed that reduced, mutated or deleted 3′ kappaenhancers not only lowered expression levels, but also decreased thelevel of somatic hypermutations. Currently, it cannot be determinedwhether the 3′ kappa enhancer is involved in SHM processes, expressionregulation or both (review V. H. Odegard et al. (2006), Nat. Rev.Immunol. 6:573; M. Inlay et al. (2002), Nat. Immunol. 3:463).

Detailed expression studies using engineered variants of the 3′ kappaenhancer indicated that a 50 nucleotide region is sufficient to driveexpression. However for proper expression a reduced sequence of 145nucleotides is preferred (EP04690251; K. B. Meyer et al. (1990), NucleicAcids Res. 18(19):5609-15).

Thus, the invention in one aspect provides a nucleic acid for insertioninto the genome of a non human animal that is an expression cassette forthe expression of a desired proteinaceous molecule in cells developinginto mature B cells during a certain stage of development, the cassettecomprising means for preventing silencing of expression of the desiredproteinaceous molecule after introduction into a host cell, and meansfor timing expression of the desired proteinaceous molecule with thedesired developmental stage of the host cell.

An expression cassette is defined as a nucleic acid that has beenprovided with means for introduction into the genome of a host cell,such as sequences which allow for homologous recombination with acertain site in the genome. Usually the nucleic acid will be DNA,typically double stranded. Typically the expression cassette will beprovided to the cell in a vector from which it is transferred to thegenome of the cell. The expression cassette further comprises allelements necessary for expression of the gene in a host cell, althoughin certain embodiments some of such elements may be present on a secondnucleic acid to be introduced, whereby these elements act in trans.Elements necessary for expression in a host cell include promoters,enhancers and other regulatory elements. Only those elements arenecessary that are not provided by the host cell.

The expression of the gene of interest should not be silenced in thegenome of the host cell, especially not in the development stage whereexpression is required. This can be done by various means, such asinsertion into the endogenous locus or by providing the cassette withnucleic acid elements that prevent silencing (Kwaks et al. (2006),Trends Biotechnol. 24(3):137-142, which is incorporated herein byreference). It is preferred that the expression cassette is inserted ina locus that is not silenced in the host cells (EP 01439234; which isincorporated herein by reference).

The means for prevention of silencing comprise STabilizingAnti-Repression-sequences (STAR®-sequences) and Matrix AttachmentRegions (MARs). A STAR sequence is a nucleic acid sequence thatcomprises a capacity to influence transcription of genes in cis.Typically, although not necessarily, a STAR sequence does not code byitself for a functional protein element. In one embodiment one STARelement is used. Preferably, however, more than one STAR element isused. In a particularly preferred embodiment an expression cassettedescribed herein is provided with two STAR sequences; one STAR sequenceat the 5′ side of the coding sequence of the immunoglobulin gene and oneSTAR sequence at the 3′ side of the coding sequence of theimmunoglobulin gene. MARs are DNA sequences that are involved inanchoring DNA/chromatin to the nuclear matrix and they have beendescribed in both mammalian and plant species. MARs possess a number offeatures that facilitate the opening and maintenance of euchromatin.MARs can increase transgene expression and limit position-effects.

Expression from the cassette should only occur during a certain periodin the development of a cell, in particular a developing B cell, more inparticular a B cell in a transgenic non-human animal, in particular amouse. In this particular case the developmental period is chosen suchthat the expression of the gene from the cassette (typically a light- orheavy chain-like polypeptide) does not significantly interfere with thenormal differentiation and/or maturation of the cell and whenapplicable, allows for pairing of the polypeptide chain produced withits counterpart.

This may, in one embodiment, be achieved by providing a nucleic aciddescribed herein, wherein the means for timing expression is a promoterof which the activity is essentially limited to the certain stage ofdevelopment. In a developing B cell, which, e.g., after immunization ismaturing and/or differentiating, the expression of the gene of interest,when it is one of the polypeptide chains of an immunoglobulin, must notinterfere (significantly) with the maturation and/or differentiation andit needs to be timed such that the resulting polypeptide can pair withits counterparts. Therefore, provided is a nucleic acid described hereinwherein the certain stage starts at a stage immediately preceding orcoinciding with the onset of the expression of light chain molecules bythe cells at a certain stage of development into a mature B cell. Thismay be achieved by selecting a promoter which is active only during thesuitable period. Such a promoter may be a CD19 promoter, the Ig-αpromoter, the Ig-β promoter, the μhc (all genes) promoter, the Vkpromoter or analogues or homologues thereof.

In a specific embodiment, the promoter as disclosed above does not drivethe expression of the gene of interest directly. Instead it drives theexpression of a gene of which the product activates in trans theexpression of the gene of interest. Such an activating gene may be agene encoding a so-called Cre recombinase or Cre-like protein. Theexpression cassette for the gene of interest may, e.g., be provided witha sequence that inhibits expression of the gene of interest. Thesequence can be removed by the action of the Cre recombinase, which isunder control of the desired promoter (active during the proper stage ofdevelopment). In this embodiment a set of expression cassettes isrequired.

Therefore, provided is a set of nucleic acids that are expressioncassettes, wherein one nucleic acid comprises an expression cassetteencoding a Cre-like protein under control of a promoter active duringthe desired stage of development of the host cell and the second nucleicacid comprises a sequence encoding a desired proteinaceous moleculeunder control of a constitutive promoter which can be activated by theaction of a Cre-like protein. The activation is preferably achieved byremoval of a stop sequence flanked by loxP sites. The Cre/lox system isdescribed in detail in Rajewsky et al. (1996), J. Clin. Invest.98:600-603, which is incorporated herein by reference. Such systems arereviewed in F. T. Wunderlich (2004), “Generation of inducible Cresystems for conditional gene inactivation in mice,”Inauguraldissertation zur Erlangung des Doktorgrades derMathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln;on the World Wide Web atdeposit.ddb.de/cgi-bin/doksery?idn=97557230x&dok_var=d1&dok_ext=pdf&filename=97557230x.pd,which is incorporated herein by reference.

Further provided is a transgenic non-human animal that has been providedwith an expression cassette hereof, wherein the desired proteinaceousmolecule is a polypeptide chain of an immunoglobulin. A preferredpolypeptide chain is a light chain. A more preferred polypeptide is agermline or germline-like light chain. A most preferred polypeptide isencoded by the immunoglobulin kappa variable 1-39 (IGKV1-39, also knownas O12) gene segment, preferably the rearranged germline kappa lightchain IGKV1-39*01/IGKJ1*01 (nomenclature according to the IMGT database,at [worldwideweb].imgt.org).

In certain embodiments, the polypeptide chain is rendered essentiallyincapable of rearrangement and/or of excluded of any sequencemodification such as normally operating on Ig during the process of Bcell affinity maturation. Therefore, provided is a transgenic non-humananimal that has been provided with an expression cassette describedherein, wherein the rearrangement and/or sequence modifications areprevented by the absence of elements at least partially responsible forsomatic hypermutation such as, for example, the MoEκi enhancer.

A preferred expression cassette described herein comprises means forprevention of silencing. In one embodiment, the means for prevention ofsilencing are means for insertion into a locus in the genome of the hostcell that is resistant to silencing. The means for insertion arepreferably means for homologous recombination into the site resistant tosilencing. A preferred locus when the non-human animal is a mouse is therosa-locus.

A further preferred expression cassette described herein comprises in5′-3′ direction: a Vκ promoter, a mouse leader, a human V gene,optionally a MoEκi enhancer, a rat constant region (Cκ) and optionally a(truncated) MoEκ3′ enhancer.

Yet a further preferred expression cassette described herein comprisesin 5′-3′ direction: a Vκ promoter, a human leader, a human V gene,optionally a MoEκi enhancer, a rat constant region (Cκ) and optionally a(truncated) MoEκ3′ enhancer.

Certain antibodies produced as described herein may be used in humantherapeutics and diagnostics. Thus, provided is a method for producing adesired antibody comprising exposing a non-human mammal described hereinto an antigen such that an antibody response is induced and isolatingthe antibodies specific for the antigen.

In certain embodiments, provided are methods for producing a desiredantibody comprising exposing a non-human mammal described herein to anantigen such that an antibody response is induced and isolating cellsproducing such antibodies, culturing and optionally immortalizing thecells and harvesting the antibodies.

In certain embodiments, provided is a method for producing a desiredantibody comprising exposing a non-human mammal described herein to anantigen such that an antibody response is induced and isolating anucleic acid encoding at least part of such an antibody, inserting thenucleic acid or a copy or a derivative thereof in an expression cassetteand expressing the antibody in a host cell.

The methods for producing antibodies from transgenic mice are known to aperson skilled in the art. Particularly preferred are methods forproduction of mixtures of antibodies from one cell, whereby the nucleicacids encoding these antibodies have been derived from mice describedherein.

These so-called oligoclonics are disclosed in WO04106375 and WO05068622,which are incorporated herein by reference.

Described herein are transgenic non-human mammals, preferably mice,capable of generating specific and high affinity hybrid mouse-humanantibodies with preferably human immunoglobulin light chain variable(VL) regions in or near germline configuration and preferably murineimmunoglobulin heavy chain variable (VH) regions that may haveaccumulated somatic mutations during the process of antigen-drivenaffinity maturation. It is envisaged that the murine VH regions of thehybrid antibodies may be subjected to humanization procedures to yieldmAbs that have reduced immunogenicity when applied in humans based ongermline or near-germline VL regions and murine VH regions that havebeen humanized.

In particular, we have shown that transgenic mice that harbor a DNAexpression construct that encodes a rearranged human VL region under thecontrol of cis-acting genetic elements that provide timely and regulatedexpression of the transgene on a significant proportion of B cellsduring B cell development, yet lack elements that direct the somatichypermutation machinery to the transgene, are capable of generatingspecific and high affinity mouse-human hybrid antibodies withessentially unmutated L chains. It is shown that the rearranged humantransgene is capable of pairing with a diversity of endogenous murineimmunoglobulin H chains to form mouse-human hybrid immunoglobulinsexpressed on the surface of B cells and to sufficiently facilitatemurine B cell development to obtain a sizeable and diverse peripheral Bcell compartment.

In certain embodiments, the transgene expression construct harbors thecoding sequences of a human rearranged L chain V region under thecontrol of a human VL promoter to direct B-cell specific expression. Inaddition, the construct harbors the murine 3′ Ck enhancer sequence for Bcell specific and inducible and high level expression of the transgene.Furthermore, the construct is designed to lack regulatory elements thatfacilitate the recruitment of the somatic hypermutation machinery to thetransgene, such as the intron enhancer and the 3′ C-kappa enhancer.

In a related embodiment, the rearranged human VL gene is inserted in themurine Rosa26 locus by site-specific integration. The Rosa26 locus isuseful in the context of the “targeted transgenesis” approach forefficient generation of transgenic organisms (such as mice) with apredictable transgene expression pattern.

In certain embodiments, the rearranged human VL region is selected forits capacity to pair with many different murine VH genes so as to ensurethe generation of a population of B cells with a diverse VH generepertoire. A method of obtaining such VL regions comprises amplifying arepertoire of rearranged VH genes from the B cells of mice and arepertoire of human rearranged germline VL regions from the B cells ofhumans and cloning them into phagemid display vectors to prepare diverselibraries of hybrid immunoglobulins in bacteria. By nucleotide sequenceanalysis of collections of unselected and antigen-selected VH/VL pairs,human germline VL genes that pair with many different murine VH genesare identified. A collection of human germline VL genes with thiscapacity is described.

In one embodiment, it is shown that upon immunization with antigen, theB cells are capable of mounting an immune response, leading to thegeneration of B cells that secrete hybrid antibodies with highspecificity and affinity. The V regions encoding these antibodies arecharacterized by the human transgenic light chain that harbors no orvery few mutations and a murine heavy chain that harbors a variablenumber of mutations introduced by the somatic hypermutation machinery.

In a related embodiment, strategies to obtain high affinity hybridmonoclonal antibodies from the transgenic mice by hybridoma and displaytechnologies are contemplated as well as procedures to humanize themurine VH regions to obtain less immunogenic antibodies for applicationin humans.

In one embodiment, provided is an immunoglobulin L chain transgeneconstruct comprising DNA sequences that encode a human immunoglobulin VLregion in combination with a light chain constant region (CL) of ananimal immunoglobulin protein, which sequences are operably linked totranscription regulatory sequences that, when integrated in a non-humantransgenic animal, produce an Ig VL-CL polypeptide with a human VLregion that is not or marginally subject to somatic hypermutation. TheIg VL is capable of pairing with rearranged VH-CH polypeptides that aregenerated during B cell development in the non-human transgenic animal,with the VH-CH polypeptides retaining the capacity to undergo somatichypermutation upon stimulation. The CL region may be of any animalspecies and is generally capable of pairing with the CH regions of thenon-human transgenic animal.

Also included is the use of a transgene construct as above in producinga transgenic non-human animal capable of the production of hybridantibodies consisting of VL-CL polypeptides and VH-CH polypeptides inwhich the VL region is of human origin and the CL, VH and CH may be ofany animal species, including human. Upon immunization, these transgenicanimals are capable of generating high affinity antibodies encoded bysomatically hypermutated VH genes and essentially non-mutated VL genesencoded by the transgene.

In another aspect, provided is a process for the production of atransgenic non-human animal capable of the production of hybridantibodies in response to antigenic challenge, comprising functionallydisrupting the endogenous immunoglobulin light chain locus and insertinginto the animal genome a transgene construct of the invention.

Included is the use of animals obtainable by this process in theproduction of B cells that produce immunoglobulin having human VL lightchain. In another aspect of the invention there is provided a processfor the production of B cells that produce immunoglobulin having humanVL and binding to a selected antigen, comprising challenging an animalobtainable by a process as above with the antigen and screening for Bcells from the animal that bind the antigen. Further included is B cellsobtainable by this process and hybridomas obtainable by immortalizingsuch B cells, e.g., hybridomas obtained by fusing B cells as above withmyeloma cells. Also included is a process for producing monoclonalantibody comprising cultivating such a hybridoma. In yet a furtheraspect, provided is the use of the above B cells in producing ahybridoma or corresponding monoclonal antibody.

Described herein is a process for the production of immunoglobulinhaving human VL chain and binding to a selected antigen, comprisingchallenging an animal obtainable as above with the antigen and obtainingimmunoglobulin there from.

In one strategy, as an individual step, a rearranged VL region encodedby human germline V and J gene segments and a light chain constantregion of any animal species but preferably a murine constant region isintroduced into the mouse germ line. The transgene DNA may be introducedinto the pronuclei of fertilized oocytes or embryonic stem cells. Theintegration may be random or homologous depending on the particularstrategy to be employed. For example, the VL transgene may be introducedby random insertion, resulting in mice that bear one or multiple copiesof the transgene in the genome. Alternatively, the human VL transgenemay be targeted to a specific genomic locus using site-specificrecombination as described in the art.

In certain embodiments, the VL transgene is targeted to the murineROSA26 locus which is a suitable integration site allowing strong andpredictable expression of inserted transgenes (European Patent Officedocument EP 1,439,234 A1, the contents of which are incorporated hereinby this reference). The targeting vector allows insertion of a singlecopy of a gene expression cassette, thus avoiding modulation oftransgene expression by the arrangement of multiple copies. By choosingthe autosomal Rosa26 locus as insertion site, the expression pattern ofthe inserted transgene in the non-human animal is predictable.Furthermore, random X inactivation and/or modulation by chromosomalposition effects are avoided. This also eliminates the need to generateand analyze multiple transgenic strains for any given transgene.Finally, the Rosa26 targeting vector for the site-specific integrationcan be used for multiple gene expression cassettes. Thus, it may beenvisaged that two or more different rearranged germline human VLregions are inserted into the Rosa26 locus to further increase thediversity of the repertoire of hybrid or human antibodies.

In another embodiment, a rearranged human VL region may be targeted tothe murine Ig kappa or lambda light chain locus so as to functionallyinactivate the endogenous locus or mice containing the rearranged humanVL region may be bred with mice that lack functional kappa or lambda Igloci or both. Thus, by using transformation, using repetitive steps orin combination with breeding, transgenic animals may be obtained whichare able to produce antibodies harboring the human VL transgene in thesubstantial absence of endogenous host immunoglobulin light chains.

In one embodiment, a human VL transgene is selected for its capacity topair with a substantial portion of murine VH regions to form a diverserepertoire of functional mouse-human hybrid antibodies expressed on thesurface of B cells. By a substantial portion of murine VH regions ismeant that the human VL pairs with at least with 0.1% of the murine VHregions generated during B cell development, more preferably with atleast 1% and most preferably with at least 10%. Methods to identifyhuman VL genes with this characteristic include randomly pairing arepertoire of human VL regions with a repertoire of murine VH regions,co-expression of VH and VL regions in appropriate eukaryotic orprokaryotic expression vectors and screening for human VL regions thatpair with a substantial portion of murine VH regions. In one embodiment,phagemid vectors may be used to direct expression of mouse-humanantibody fragments in bacterial cells or to the surface of filamentousphage and analysis of binding capacity of antibody fragments by methodsknown in the art.

In another embodiment, a human VL transgene is selected for its capacityto pair with a substantial portion of human VH regions to form a diverserepertoire of human antibodies expressed on the surface of B cells. By asubstantial portion of human VH regions is meant that the human VL pairswith at least with 0.1% of the human VH regions generated during B celldevelopment, more preferably with at least 1% and most preferably withat least 10%.

In the latter embodiment, the human VL transgenic mice are crossed withmice that harbor functional rearranged or non-rearranged human H chainimmunoglobulin loci and functionally inactivated endogenous H chain Igloci as described in the art. The functional inactivation of the twocopies of each of the three host Ig loci (heavy chain, kappa and lambdalight chain), where the host contains the human IgH and the rearrangedhuman VL transgene would allow for the production of purely humanantibody molecules without the production of host or host human chimericantibodies. Such a host strain, by immunization with specific antigens,would respond by the production of mouse B-cells producing specifichuman antibodies, which B-cells are subsequently fused with mousemyeloma cells or are immortalized in any other manner for the continuousstable production of human monoclonal antibodies. Alternatively, thepopulation of B cells is used as a source of VH regions that can beobtained by constructing cDNA libraries or by PCR amplification usingprimers for human VH regions as is known in the art.

A human rearranged VL gene is reconstructed in an appropriate eukaryoticor prokaryotic microorganism and the resulting DNA fragments can beintroduced into pronuclei of fertilized mouse oocytes or embryonic stemcells. Various constructs that direct B cell specific expression of VLtransgenes have been described in the art and have the following generalformat: a leader sequence and relevant upstream sequences to direct Bcell specific expression of the transgene, a coding sequence of a humanVL transgene, an enhancer sequence that directs B cell specific and highlevel expression of the transgene and a murine constant region gene. Ina preferred format, the enhancer is the C-kappa 3′ enhancer because itdirects high level expression in B-lineage cells, but does not recruitsomatic hypermutation when used in transgene constructs.

In one embodiment, animals, preferably mice, comprising one or multiplecopies of the transgene in the genome are isolated and analyzed forstable expression. Animals are selected that show stable expression ofthe transgene over longer periods of time, preferably in B-cells. Ifrequired, different animal lines comprising independent insertions ofone or multiple copies of the transgene, preferably on differentchromosomes, are crossed to obtain animals with different insertions ofone or multiple copies of the transgene to increase expression of thetransgene in animals, preferably in B-cells.

Further provided is progeny of a transgenic non-human animal describedherein, the progeny comprising, at least in its B-cell lineage, a heavy-or light chain encoding sequence together with a means that renders thesequence resistant to DNA rearrangements and/or somatic hypermutations.

Further provided is progeny of a transgenic non-human animal describedherein, the progeny comprising an expression cassette for the expressionof a desired proteinaceous molecule in cells during a certain stage ofdevelopment in cells developing into mature B cells.

In addition, provided is a cell that is isolated from a transgenicnon-human animal described herein, the cell comprising a heavy- or lightchain encoding sequence together with a means that renders the sequenceresistant to DNA rearrangements and/or somatic hypermutations. Inaddition, provided is a cell that is isolated from a transgenicnon-human animal described herein, the cell comprising an expressioncassette for the expression of a desired proteinaceous molecule in cellsduring a certain stage of development in cells developing into mature Bcells. A cell described herein, preferably an antibody-producing B-cellor a cell that is capable of differentiating or maturating into anantibody-producing B-cell, can be used for in vitro production ofantibodies, as is known to the skilled person, for example, from Gascanet al. 1991, J. Exp. Med. 173:747-750. Methods for immortalization of acell described herein are known in the art and include the generation ofhybridomas, for example, by fusion with a myeloma cell, transformationwith Epstein Barr Virus; expression of the signal transducer ofactivation and transcription (STAT), activation via CD40 and IL4receptor signaling, and/or expression of Bcl6 (Shvarts et al. 2002,Genes Dev. 16: 681-686).

In a separate step, the mouse endogenous Kappa and Lambda light chainloci are rendered essentially non-functional such that at least themajority of B cells in the transgenic mice bear Ig receptors thatcontain the transgenic human VL region. Inactivation of the endogenousmouse immunoglobulin loci is achieved by targeted disruption of theappropriate loci by homologous recombination in mouse embryonic stemcells. The targeted disruption comprises alteration of the genomicsequence such that substantially no functional endogenous mouseimmunoglobulin Kappa and/or Lambda light chain is produced. The term“substantially no functional endogenous mouse immunoglobulin” indicatesthat the endogenous Kappa and/or Lambda light chain loci arefunctionally silenced such that the level of functional proteinexpression of the endogenous Kappa and/or Lambda light chain loci,preferably the endogenous Kappa light chain locus, is reduced to about20% of the level of expression in a reference mouse, more preferred toabout 10%, more preferred to about 5%, more preferred to about 2% andmore preferred to about 1%. In a most preferred embodiment, the level offunctional protein expression of the endogenous Kappa and/or Lambdalight chain loci is reduced to 0%. The level of functional proteinexpression can be determined by means known to the skilled person,including western blotting and pairing with a mouse heavy chain. Thereference mouse is a mouse in which the endogenous Kappa and/or Lambdalight chain loci is not disrupted. The alteration comprises mutationand/or deletion of gene sequences that are required for functionalexpression of the endogenous immunoglobulin genes. Alternatively, thealteration comprises insertion of a nucleic acid into the endogenousmouse immunoglobulin Kappa and/or Lambda light chain loci such that thefunctional expression of the endogenous immunoglobulin genes is reduced.In one embodiment, the nucleic acid comprises a silencing elementresulting in transcriptional silencing of the endogenous immunoglobulingene. In a further embodiment, or in addition, the nucleic acidcomprises a sequence that disrupts splicing and/or translation of theendogenous immunoglobulin gene, for example, by introducing an exon thatrenders a frame shift in the coding sequence, or that comprises apremature stop codon. In each case chimeric animals are generated whichare derived in part from the modified embryonic stem cells and arecapable of transmitting the genetic modifications through the germ line.The mating of mouse strains with human immunoglobulin loci to strainswith inactivated mouse loci yields animals which produce antibodiescomprising essentially only human light chains.

A construct for homologous recombination is prepared by means known inthe art and any undesirable sequences are removed, e.g., procaryoticsequences. Any convenient technique for introducing a construct forhomologous recombination into a target cell may be employed. Thesetechniques include spheroplast fusion, lipofection, electroporation,calcium phosphate-mediated DNA transfer or direct microinjection. Aftertransformation or transfection of the target cells, target cells areselected by means of positive and/or negative markers, for example, byneomycin resistance and/or acyclovir and/or gancyclovir resistance.Those cells which show the desired phenotype may then be furtheranalyzed by restriction analysis, electrophoresis, Southern analysis,PCR, or the like. By identifying fragments which show the presence ofthe lesion(s) at the target locus, cells in which homologousrecombination has occurred to inactivate a copy of the target locus areidentified.

Furthermore, it is shown that upon immunization, the murine and human VHregions in the afore-mentioned transgenic mice but not the VL regionsare capable of undergoing somatic hypermutations to generate highaffinity antibodies. Advantageously, these antibodies encoded bygermline VL regions are predicted to contribute to lower immunogenicitywhen applied in humans and result in more stable antibodies that areless prone to aggregation and thus safer for therapeutic use in humans.

MAbs derived from the afore-mentioned non-human transgenic animals orcells all share the same identical human VL regions. It has beendescribed that mAbs that share the same identical VL region may beco-expressed in a single clonal cell for the production of mixtures ofrecombinant antibodies with functional binding sites (see theincorporated WO04106375 and WO05068622). Thus, provided is a platformfor the generation of specific and high affinity mAbs that constitutethe basis for mixtures of mAbs produced by clonal cells.

It is preferred that mAbs derived from the afore-mentioned non-humantransgenic animals or cells are directed against cellular targets.Preferred targets are human surface-expressed or soluble proteins orcarbohydrate molecules. Further preferred targets are surface-expressedproteins or carbohydrate molecules that are expressed on the surface ofbacteria, viruses, and other pathogens, especially of humans.

More specifically, preferred targets include cytokines and chemokines,including but not limited to InterLeukin 1beta (IL1beta), IL2, IL4, IL5,IL7, IL8, IL12, IL13, IL15, IL18, IL21, IL23 and chemokines such as, forexample, CXC chemokines, CC chemokines, C chemokines (or γ chemokines)such as XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β), and CX3Cchemokines. Further included as preferred targets are receptor moleculesof the cytokines and chemokines, including type I cytokine receptorssuch as, for example, the IL-2 receptor, type II cytokine receptors suchas, for example, interferon receptors, immunoglobulin (Ig) superfamilyreceptors, tumor necrosis factor receptor family including receptors forCD40, CD27 and CD30, serine/threonine-protein kinase receptors such asTGF beta receptors, G-protein coupled receptors such as CXCR1-CXCR7, andtyrosine kinase receptors such as fibroblast growth factor receptor(FGFR) family members, EGF receptor family members including erbB1(EGF-R; HER1), erbB2, (HER2), erbB3 (HER3), and erbB4 (HER4), insulinreceptor family members including IGF-R1 and IGF-RII, PDGF receptorfamily members, Hepatocyte growth factor receptor family membersincluding c-Met (HGF-R), Trk receptor family members, AXL receptorfamily members, LTK receptor family members, TIE receptor familymembers, ROR receptor family members, DDR receptor family members, KLGreceptor family members, RYK receptor family members, MuSK receptorfamily members, and vascular endothelial growth factor receptor (VEGFR)family members.

Further preferred targets are targets that are over-expressed orselectively expressed in tumors such as, for example, VEGF, CD20, CD38,CD33, CEA, EpCAM, PSMA, CD54, Lewis Y, CD52, CD40, CD22, CD51/CD61,CD74, MUC-1, CD38, CD19, CD262 (TRAIL-R2), RANKL, CTLA4, and CD30;targets that are involved in chronic inflammation such as, for example,CD25, CD11a, TNF, CD4, CD80, CD23, CD3, CD14, IFNgamma, CD40L, LD50,CD122, TGFbeta and TGFalpha.

Preferred surface-expressed proteins or carbohydrate molecules that areexpressed on the surface of bacteria, viruses, and other parasiticpathogens, especially of humans, include surface markers of influenza Aand B viruses such as hemagglutinin (HA) and neuraminidase (NA),filoviruses such as Ebola virus, rabies, measles, rubella, mumps,flaviviruses such as Dengue virus types 1-4, tick-borne encephalitisvirus, West Nile virus, Japanese encephalitis virus, and Yellow fevervirus, Paramyxoviruses including Paramyxovirus such as Parainfluenza 1,3, Rubulavirus such as Mumpsvirus and Parainfluenza 2, 4, Morbillivirus,and Pneumovirus such as Respiratory syncytial virus, Vaccinia, smallpox, coronaviruses, including Severe Acute Respiratory Syndrome (SARS)virus, hepatitis virus A, B and C, Human Immunodeficiency Virus, Herpesviruses, including cytomegalovirus, Epstein Barr virus, Herpes simplexvirus, and Varicella zoster virus, parvoviruses such as, for example,B19; Legionella pneumophila; Listeria monocytogenes; Campylobacterjejuni; Staphylococcus aureus; E. coli O157:H7; Borrelia burgdorferi;Helicobacter pylori; Ehrlichia chaffeensis; Clostridium difficile;Vibrio cholera; Salmonella enterica Serotype Typhimurium; Bartonellahenselae; Streptococcus pyogenes (Group A Strep); Streptococcusagalactiae (Group B Strep); Multiple drug resistant S. aureus (e.g.,MRSA); Chlamydia pneumoniae; Clostridium botulinum; Vibrio vulnificus;Parachlamydia pneumonia; Corynebacterium amycolatum; Klebsiellapneumonia; Linezolid-resistant enterococci (E. faecalis and E. faecium);and Multiple drug resistant Acinetobacter baumannii.

Most preferred targets are IL-6 and its receptor, IL-6Ralpha,glycoprotein-denominated gp130, RSV, especially the surface proteins F,G and SH and non-structural proteins such as N and M, and receptortyrosine kinases, in particular erbB1 (EGF-R; HER1), erbB2, (HER2),erbB3 (HER3), erbB4 (HER4), IGF-R1 and IGF-RII, c-Met (HGF-R).

Therefore, provided is a platform for the generation of specific andhigh affinity mAbs against the above mentioned targets that constitutethe basis for mixtures of mAbs produced by clonal cells. In certainembodiments, the specific and high affinity mAbs comprise mAbs that aredirected against different epitopes on at least one of the targets. In afurther preferred embodiment, the specific and high affinity mAbscomprise mAbs that are directed against different targets, such as, forexample, one or more members of the EGF-receptor family, including erbB1(EGF-R; HER1), erbB2, (HER2), erbB3 (HER3) and erbB4 (HER4).

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (3rd edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)), which isincorporated herein by reference. The nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: A topology map of the annealing locations of mouse specific VHprimers and the position of required restriction sites that areintroduced by overhanging sequences at the 3′ end of primers.

FIG. 2: PCR amplification steps (Amplification, Intermediate and Siteintroduction). The location and names of the mouse VH amplificationprimers (and mixtures of primers) are indicated per step.

FIG. 3: Topology of the MV1043 vector. This vector is used for thecloning of human or murine VH fragments. O12 (IGKV1-39) is indicated asthe VL gene. Products of this vector in combination with helper phagesin E. coli cells allow the generation of phages that display Fabfragments on the surface of the phage particles as a fusion product tothe g3 protein and presence of the vector in the phage as the geneticcontent (F1 ORI).

FIG. 4: The topology of the mouse Ckappa locus downstream of theJ-segments. Both enhancers and Ckappa region are indicated. The lowerarrow indicates the region that is removed in order to silence thelocus.

FIG. 5: The topology of the mouse C-lambda locus. All three activeV-regions are indicated (Igl-V1, V2 and V3) as are the J-segments(Igl-J1, Igl-J2, Igl-J3, Igl-J4 and the pseudo segment Igl-J3p) andconstant regions (Igl-C1, Igl-C2, Igl-C3 and Igl-C4). The regions thatare deleted in order to silence the locus are indicated by deletionmarkers. These deletions include all active V genes (1, 2 and 3) and theintergenic segment between V2 and V3.

FIG. 6: Construct topology of IGKV1-39/J-Ck with an intron located inthe leader open reading frame (ORF).

FIG. 7: Construct topology of IGLV2-14/J-Ck with an intron located inthe leader open reading frame (ORF).

FIG. 8: Construct topology of VkP-IGKV1-39/J-Ck (VkP-O12). The promoteroriginates from the IGKV1-39 gene and is placed directly in front of therequired elements for efficient transcription and translation.Intergenic sequences (including the enhancers) are derived from mice andobtained from BAC clones. The C-kappa sequence codes for the kappaconstant region of rat.

FIG. 9: Construct topology of VkP-IGLV2-14/J-Ck (VkP-2a2). The promoteroriginates from the IGKV1-39 gene and is placed directly in front of therequired elements for efficient transcription and translation.Intergenic sequences (including the enhancers) are derived from mice andobtained from BAC clones. The C-kappa sequence codes for the kappaconstant region of rat.

FIG. 10: Construct topology of VkP-IGKV1-39/J-Ck-Δ1 (VkP-O12-del1) isidentical to VkP-IGKV1-39/J-Ck from FIG. 9 except that the intronenhancer region is removed.

FIG. 11: Construct topology of VkP-IGKV1-39/J-Ck-Δ2 VkP-O12-del2) isidentical to VkP-IGKV1-39/J-Ck-Δ1 from FIG. 10 except that a large pieceof the intergenic region between the Ck gene and 3′ enhancer is deleted.In addition, the 3′ enhancer is reduced in size from 809 bp to 125 bp.

FIG. 12: Overview of the sequences used or referred to in thisapplication: Human germline IGKV1-39/J DNA (SEQ ID NO:84); humangermline IGKV1-39/J Protein (SEQ ID NO:85); human germline IGLV2-14/JDNA (SEQ ID NO:86); human germline IGLV2-14/J Protein (SEQ ID NO:87);Rat IGCK allele a DNA (SEQ ID NO:88); Rat IGCK allele a protein (SEQ IDNO:89); IGKV1-39/J-Ck (SEQ ID NO:90); IGLV2-14/J-Ck (SEQ ID NO:91);VkP-IGKV1-39/J-Ck (SEQ ID NO:92); VkP-IGKV1-39/J-Ck-Δ1 (SEQ ID NO:93);VkP-IGKV1-39/J-Ck-Δ2 (SEQ ID NO:94); VkP-IGLV2-14/J-Ck (SEQ ID NO:95);pSELECT-IGKV1-39/J-Ck (SEQ ID NO:96); pSelect-IGLV2-14/J-Ck (SEQ IDNO:97); MV1043 (SEQ ID NO:98); and MV1057 (SEQ ID NO:99).

FIGS. 13A-C: Generation of Rosa26-IgVk1-39 KI allele. FIG. 13A Schematicdrawing of the pCAGGS-IgVK1-39 targeting vector. FIG. 13B Nucleotidesequence of the pCAGGS-IgVK1-39 targeting vector (SEQ ID NO:100). FIG.13C Targeting strategy.

FIGS. 14A-C: FIG. 14A Southern blot analysis of genomic DNA of ES clonescomprising an insertion of the pCAGGS-IgVK1-39 targeting vector. GenomicDNA of four independent clones was digested with AseI and probed with5e1 indicating the 5′-border of the targeting vector. All clonescomprise a correct insertion of the targeting vector at the 5′ end. FIG.14B Southern blot analysis of genomic DNA of ES clones comprising aninsertion of the pCAGGS-IgVK1-39 targeting vector. Genomic DNA of fourindependent clones was digested with MscI and probed with 3e1 indicatingthe 3′-border of the targeting vector. All clones comprise a correctinsertion of the targeting vector at the 3′ end. FIG. 14C Southern blotanalysis of genomic DNA of ES clones comprising an insertion of thepCAGGS-IgVK1-39 targeting vector. Genomic DNA of four independent cloneswas digested with BamHI and probed with an internal Neo probe indicatingthe 5′-border of the targeting vector. All clones comprise a correct,single insertion of the targeting vector.

FIGS. 15A-C: Generation of Rosa26-IgV12-14 KI allele. FIG. 15A Schematicdrawing of the pCAGGS-IgVL2-14 targeting vector. FIG. 15B Nucleotidesequence of the pCAGGS-IgVL2-14 targeting vector containing the CAGGSexpression insert (SEQ ID NO:101) based on the rearranged germlineIGLV2-14/J V lambda region (IGLV2-14/J-Ck). FIG. 15C Targeting strategy.

FIGS. 16A-C: Epibase® profile of IGKV1-39 residues 1-107 (SEQ ID NO:85).

FIG. 16A displays the binding strength for DRB1 allotypes, while FIG.16C displays the binding strength for DRB3/4/5, DQ and DP allotypes. Thevalues in the figure represent dissociation constants (Kds) and areplotted on a logarithmic scale in the range 0.01 μM-0.1 μM (very strongbinders may have run off the plot). For medium binding peptides,qualitative values are given only, and weak and non-binders are notshown. Values are plotted on the first residue of the peptide in thetarget sequence (the peptide itself extends by another nine residues).Importantly, only the strongest binding receptor for each peptide isshown: cross-reacting allotypes with lower affinity are not visible inthis plot. The strongest binding receptor is indicated by itsstereotypic name. Finally, any germline-filtered peptides are plottedwith a lighter color in the epitope map (in this case, no non-selfepitopes were found). FIG. 16B shows the HLA binding promiscuity forevery decameric peptide (Y-axis: the number of HLA allotypes recognizingcritical epitopes in each of the peptides starting at the indicatedresidue shown on the X-axis). The promiscuity is measured as the numberof allotypes out of the total of 47 for which the peptide is a criticalbinder. White columns refer to self-peptides, and black columns (absenthere) to non-self peptides.

FIG. 17: Epitope map of IGKV1-39 showing the presence of peptide binderspredicted in the sequence of IGKV1-39 by serotype in the 15-mer format.Each 15-mer is numbered as indicated in the top of the figure. The fullsequence of the corresponding 15-mer is listed in Table 7. Black boxesindicate the presence of one or more critical self-epitopes in the15-mer for the serotype listed on the left. Critical epitopes areoperationally defined as strong or medium DRB1 binders and strongDRB3/4/5 or DP or DQ binders.

FIGS. 18A-B: Constitutive knock-out (KO) of the Ig kappa locus. FIG. 18ATargeting strategy. FIG. 18B Schematic drawing of the pIgKappa targetingvector.

FIGS. 19A-B: Constitutive KO of the Ig lambda locus. FIG. 19A First stepof the targeting strategy. FIG. 19B Second step of the targetingstrategy.

FIGS. 20A-C: Schematic drawing of targeting vectors. FIG. 20A pVkP-O12(VkP-IGKV1-39/J-Ck); FIG. 20B pVkP-O12-del1 (VkP-IGKV1-39/J-Ck-Δ1); FIG.20C pVkP-O12-del2 (VkP-IGKV1-39/J-Ck-Δ2).

FIGS. 21A-C: Targeting strategies for insertion of transgene into theRosa26 locus by targeted transgenesis using RMCE. FIG. 21A VkP-O12(VkP-IGKV1-39/J-Ck); FIG. 21B VkP-O12-del1 (VkP-IGKV1-39/J-Ck-Δ1); FIG.21C VkP-O12-del2 (VkP-IGKV1-39/J-Ck-Δ2).

FIG. 22: Topology of the MV1057 vector. Replacing the indicated stufferfragment with a VH fragment yields an expression vector that can betransfected to eukaryotic cells for the production of IgG1 antibodieswith light chains containing an O12 (IGKV1-39) VL gene.

FIG. 23: Lack of transgenic human Vk1 light chain expression in non-Bcell populations of the spleen.

FIG. 24: Transgenic human Vk1 light chain is expressed in all B cellpopulations of the spleen.

FIG. 25: Transgenic human Vk1 light chain is expressed in B1 cells ofthe peritoneal cavity.

FIGS. 26A-B: Transgenic human Vk1 light chain is not expressed in pro-and pre-B cells but in the immature and recirculating populations Bcells in the bone marrow. FIG. 26A Gating of bone marrow cells. FIG. 26BHistograms of transgene expression with overlay from one WT control.

FIG. 27: Transgenic human Vk1 light chain is directly correlated withendogenous light chain and IgM expression in circulating B cells in theblood.

FIG. 28: Parameters of stability for stable clones containing thegermline IGKV1-39 gene.

FIG. 29A-B: Antibody mixtures used for staining of lymphocytepopulations. BM=bone marrow, PC=peritoneal cavity, PP=Peyer's patches.

DETAILED DESCRIPTION OF THE INVENTION Examples Example 1: Human LightChain V-Gene Clones

This example describes the rationale behind the choice of two humanlight chain V-genes, one gene of the kappa type and one gene of thelambda type, that are used as a proof of concept for light chainexpressing transgenic mice. De Wildt et al. 1999 (de Wildt et al.(1999), J. Mol. Biol. 285(3):895) analyzed the expression of human lightchains in peripheral IgG-positive B-cells. Based on these data, IGKV1-39(O12) and IGLV2-14 (2a2) were chosen as light chains as they were wellrepresented in the B-cell repertoire. The J-segment sequence of thelight chains has been chosen based upon sequences as presented inGenBank ABA26122 for IGKV1-39 (B. J. Rabquer, S. L. Smithson, A. K.Shriner and M. A. J. Westerink) and GenBank AAF20450 for IGLV2-14 (O.Ignatovich, I. M. Tomlinson, A. V. Popov, M. Bruggemann and G. J.Winter, J. Mol. Biol. 294 (2):457-465 (1999)).

All framework segments are converted into germline amino acid sequencesto provide the lowest immunogenicity possible in potential clinicalapplications.

Example 2: Obtaining Mouse Heavy Chain V-Genes that Pair with HumanIGKV1-39 Gene Segment to Form Functional Antibody Binding Sites

This example describes the identification of mouse heavy chain V-genesthat are capable of pairing with a single, rearranged human germlineIGKV1-39/J region. A spleen VH repertoire from mice that were immunizedwith tetanus toxoid was cloned in a phage display Fab vector with asingle human IGKV1-39-C kappa light chain and subjected to panningagainst tetanus toxoid. Clones obtained after a single round of panningwere analyzed for their binding specificity. The murine VH genesencoding tetanus toxoid-specific Fab fragments were subjected tosequence analysis to identify unique clones and assign VH, DH and JHutilization.

Many of the protocols described here are standard protocols for theconstruction of phage display libraries and the panning of phages forbinding to an antigen of interest and described in Antibody PhageDisplay: Methods and Protocols (editor(s): Philippa M. O'Brien andRobert Aitken).

Immunizations

BALB/c mice received one immunization with tetanus toxoid and wereboosted after six weeks with tetanus toxoid.

Splenocyte Isolation

Preparation of spleen cell suspension. After dissection, the spleen waswashed with PBS and transferred to a 60 mm Petri dish with 20 ml PBS. Asyringe capped with 20 ml PBS and a G20 needle was used to repeatedlyflush the spleen. After washing the flushed cells with PBS, the cellswere carefully brought into suspension using 20 ml PBS and left on abench for five minutes to separate the splenocytes from the debris andcell clusters. The splenocytes suspension was transferred on top of aFicoll-Paque™ PLUS-filled tube and processed according to themanufacturer's procedures for lymphocyte isolation (AmershamBiosciences).

RNA Isolation and cDNA Synthesis

After isolation and pelleting of lymphocytes, the cells were suspendedin TRIzol LS Reagent (Invitrogen) for the isolation of total RNAaccording to the accompanying manufacturer's protocol and subjected toreverse transcription reaction using 1 microgram of RNA, Superscript IIIRT in combination with dT20 according to manufacturer's procedures(Invitrogen).

PCR Amplification of cDNA

The cDNA was amplified in a PCR reaction using primer combinations thatallow the amplification of approximately 110 different murine V-genesbelonging to 15 VH families (Table 1; RefSeq NG_005838; Thiebe et al.1999, European Journal of Immunology 29:2072-2081). In the first round,primer combinations that bind to the 5′ end of the V-genes and 3′ end ofthe J regions were used. In the second round, PCR products that weregenerated with the MJH-Rev2 primer were amplified in order to introducemodifications in the 3′ region to enable efficient cloning of theproducts. In the last round of amplification, all PCR products wereamplified using primers that introduce a SfiI restriction site at the 5′end and a BstEII restriction site at the 3′ end (see FIGS. 1 and 2, andTable 1).

Reaction conditions for 1st round PCR: four different reactionscombining all 25 forward primers (MVH1 to MVH25, Table 1 and FIG. 2) andone reverse primer per reaction (MJH-Rev1, MJH-Rev2, MJH-Rev3 orMJH-Rev4; see Table 1 and FIG. 2). Fifty microliters PCR volumes werecomposed of 2 microliters cDNA (from RT reactions), 10 microliters 5*Phusion polymerase HF buffer, 40 nM of each of the 25 forward primers(total concentration of 1 micromolar), 1 micromolar reverse primer, 1microliter 10 mM dNTP stock, 1.25 unit Phusion polymerase and sterile MQwater. The thermocycler program consisted of a touch down program: onecycle 98° C. for 30 seconds, 30 cycles 98° C. for ten seconds, 58° C.decreasing 0.2° C. per cycle ten seconds, 72° C. 20 seconds and onecycle 72° C. for three minutes. The second round PCR program was set uponly for the products of the first PCR that contain the MJH-Rev2 primer:two different reactions combining either the ExtMVH-1 or ExtMVH-2primers (Table 1 and FIG. 2) in combination with the reverse primerExtMJH-Rev2int (Table 1 and FIG. 2). Fifty microliters PCR volumes werecomposed of 50 ng PCR product (from first PCR round), 10 microliters 5*Phusion polymerase HF buffer, 500 nM of each forward primer, 1micromolar reverse primer, 1 microliter 10 mM dNTP stock, 1.25 unitPhusion polymerase and sterile MQ water. The thermocycler programconsisted of a touch down program followed by a regular amplificationstep: one cycle 98° C. for 30 seconds, ten cycles 98° C. for tenseconds, 65° C. decreasing 1.5° C. per cycle ten seconds, 72° C. 20seconds, ten cycles 98° C. for ten seconds, 55° C. ten seconds, 72° C.20 seconds and one cycle 72° C. for three minutes. The third round PCRprogram was setup as described in FIG. 2. Fifty microliters PCR volumeswere composed of 50 ng PCR product (from earlier PCR rounds, FIG. 2), 10microliters 5* Phusion polymerase HF buffer, 1 micromolar forward primer(Table 1 and FIG. 2), 1 micromolar reverse primer, 1 microliter 10 mMdNTP stock, 1.25 unit Phusion polymerase and sterile MQ water. Theprogram consists of a touch down program followed by a regularamplification step: one cycle 98° C. for 30 seconds, ten cycles 98° C.for ten seconds, 65° C. decreasing 1.5° C. per cycle ten seconds, 72° C.20 seconds, ten cycles 98° C. for ten seconds, 55° C. ten seconds, 72°C. 20 seconds and one cycle 72° C. for three minutes. After PCRamplifications, all PCR products were gel purified using Qiaex IIaccording to the manufacturer's protocols.

Restriction Enzyme Digestions

Purified products were digested with BstEII and SfiI in two steps. First1 microgram of DNA was digested in 100 microliters reactions consistingof 10 microliters of 10* NEB buffer 3 (New England Biolabs), 1microliter 100* BSA, 12.5 unit BstEII and sterile water for six hours at60° C. in a stove. The products were purified using Qiaquick PCRPurification kit from Qiagen according to the manual instructions andeluted in 40 microliters water. Next all products were further digestedwith SfiI in 100 microliters reactions consisting of 10 microliters of10* NEB buffer 2 (New England Biolabs), 1 microliter 100* BSA, 12.5 unitSfiI and sterile water for 12 hours at 50° C. in a stove. The digestedfragments were purified by Qiaquick Gel Extraction kit following gelseparation on a 20 cm 1.5% agarose TBE plus ethidium bromide gel at 80V. 100 micrograms of the acceptor vector (MV1043, FIGS. 3 and 12) wasdigested with 50 units Eco91I in 600 microliters under standardconditions (Tango buffer) and next purified on a 0.9% agarose gel. Aftera second digestion step under prescribed conditions with 400 units SfiIin 500 microliters for 12 hours, 100 units BsrGI were added for threehours at 50° C.

Ligations

Each PCR product was ligated separately according to the followingscheme: 70 ng digested PCR products, 300 ng digested acceptor vector,100 units T4 Ligase (NEB), 1* ligase buffer in 30 microliters for 16hours at 12° C. The ligation reactions were purified withphenol/chloroform/isoamyl alcohol extractions followed by glycogenprecipitations (Sigma Aldrich #G1767) according to the manufacturer'sprotocol and finally dissolved in 25 microliters sterile water.

Transformations and Library Storage

The purified ligation products were transformed by electroporation using1200 microliters TG1 electrocompetent bacteria (Stratagene #200123) perligation batch and plated on LB carbenicillin plates containing 4%glucose. Libraries were harvested by scraping the bacteria in 50 ml LBcarbenicillin. After centrifugation at 2000 g for 20 minutes at 4° C.,the bacterial pellets were resuspended carefully in 2 ml ice cold2*TY/30% glycerol on ice water and frozen on dry ice/ethanol beforestorage at −80° C.

Library Amplification

Libraries were grown and harvested according to procedures as describedby Kramer et al. 2003 (Kramer et al. (2003), Nucleic Acids Res.31(11):e59) using VCSM13 (Stratagene) as helper phage strain.

Selection of Phages on Coated Immunotubes

Tetanus toxoid was dissolved in PBS in a concentration of 2 μg/ml andcoated to MAXISORP™ Nunc-Immuno Tube (Nunc 444474) overnight at 4° C.After discarding the coating solution, the tubes were blocked with 2%skim milk (ELK) in PBS (blocking buffer) for one hour at RT. Inparallel, 0.5 ml of the phage library was mixed with 1 ml blockingbuffer and incubated for 20 minutes at room temperature. After blockingthe phages, the phage solution was added to the tetanus toxoid-coatedtubes and incubated for two hours at RT on a slowly rotating platform toallow binding. Next, the tubes were washed ten times with PBS/0.05%TWEEN™-20 detergent followed by phage elution by an incubation with 1 ml50 mM glycine-HCl pH 2.2 ten minutes at RT on rotating wheel anddirectly followed by neutralization of the harvested eluent with 0.5 ml1 M Tris-HCl pH 7.5.

Harvesting Phage Clones

Five ml XL1-Blue MRF (Stratagene) culture at O.D. 0.4 was added to theharvested phage solution and incubated for 30 minutes at 37° C. withoutshaking to allow infection of the phages. Bacteria were plated onCarbenicillin/Tetracycline 4% glucose 2*TY plates and grown overnight at37° C.

Phage Production

Phages were grown and processed as described by Kramer et al. 2003(Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) using VCSM13 ashelper phage strain.

Phage ELISA

ELISA plates were coated with 100 microliters tetanus toxoid per well ata concentration of 2 micrograms/ml in PBS overnight at 4° C. Platescoated with 100 microliters thyroglobulin at a concentration of 2micrograms/ml in PBS were used as a negative control. Wells wereemptied, dried by tapping on a paper towel, filled completely withPBS-4% skimmed milk (ELK) and incubated for one hour at room temperatureto block the wells. After discarding the block solution, phage miniprepspre-mixed with 50 μl blocking solution were added and incubated for onehour at RT. Next five washing steps with PBS-0.05% Tween-20 removedunbound phages. Bound phages were detected by incubating the wells with100 microliters anti-M13-HRP antibody conjugate (diluted 1/5000 inblocking buffer) for one hour at room temperature. Free antibody wasremoved by repeating the washing steps as described above, followed byTMB substrate incubation until color development was visible. Thereaction was stopped by adding 100 microliters of 2 M H₂SO₄ per well andanalyzed on an ELISA reader at 450 nm emission wavelength (Table 2).Higher numbers indicate stronger signals and thus higher incidence ofspecific binding of the phage-Fab complex.

Sequencing

Clones that gave signals at least three times above the backgroundsignal (Table 2) were propagated, used for DNA miniprep procedures (seeprocedures Qiagen miniPrep manual) and subjected to nucleotide sequenceanalysis. Sequencing was performed according to the Big Dye 1.1 kitaccompanying manual (Applied Biosystems) using a reverse primer(CH1_Rev1, Table 1) recognizing a 5′ sequence of the CH1 region of thehuman IgG1 heavy chain (present in the Fab display vector MV1043, FIGS.3 and 12). Mouse VH sequences of 28 tetanus toxoid binding clones aredepicted in Table 3. The results show that the selected murine VH genesbelong to different gene families, and different individual members fromthese gene families are able to pair with the rearranged humanIGKV1-39/J VH region to form functional tetanus toxoid-specific antibodybinding sites. From the sequence analyses, it was concluded that themurine VH regions utilize a diversity of DH and JH gene segments.

Example 3: Silencing of the Mouse Kappa Light Chain Locus

This example describes the silencing of the mouse endogenous kappa lightchain locus. The endogenous kappa locus is modified by homologousrecombination in ES cells, followed by the introduction of geneticallymodified ES cells in mouse embryos to obtain genetically adaptedoffspring.

A vector that contains an assembled nucleotide sequence consisting of apart comprising the J-region to 338 bp downstream of the J5 gene segmentfused to a sequence ending 3′ of the 3′ CK enhancer is used forhomologous recombination in ES cells. The assembled sequence is used todelete a genomic DNA fragment spanning from 3′ of the JK region to just3′ of the 3′ CK enhancer. As a consequence of this procedure, the CKconstant gene, the 3′ enhancer and some intergenic regions are removed(see FIGS. 4 and 18A-B).

Construction of the Targeting Vector

A vector that received 4.5-8 kb flanking arms on the 3′ and 5′ end fusedto the deletion segment was used for targeted homologous recombinationin an ES cell line. Both arms were obtained by PCR means ensuringmaximum homology. The targeting strategy allows generation ofconstitutive KO allele. The mouse genomic sequence encompassing the Igkintronic enhancer, Igk constant region and the Igk 3′ enhancer wasreplaced with a PuroR cassette, which was flanked by F3 sites andinserted downstream of the Jk elements. Flp-mediated removal of theselection marker resulted in a constitutive KO allele. The replacementof the Igk MiEk-Igk C-Igk 3′E genomic region (approximately 10 kb) witha F3-Puro cassette (approx. 3 kb) was likely to decrease the efficiencyof homologous recombination. Therefore, the arms of homology wereextended accordingly and more ES cell colonies were analyzed aftertransfection in order to identify homologous recombinant clones.

Generation of ES Cells Bearing the Deleted Kappa Fragment

The generation of genetically modified ES cells was essentiallyperformed as described (Seibler et al. (2003), Nucleic Acids Res.February 15; 31(4):e12). See also Example 14 for a detailed description.

Generation of ES Mice by Tetraploid Embryo Complementation

The production of mice by tetraploid embryo complementation usinggenetically modified ES cells was essentially performed as described(Eggan et al., PNAS 98:6209-6214; J. Seibler et al. (2003), NucleicAcids Res. February 15; 31(4):e12; Hogan et al. (1994), Summary of mousedevelopment, Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor N.Y., pp. 253-289).

Example 4: Silencing of the Mouse Lambda Light Chain Locus

This example describes the silencing of the mouse endogenous lambdalight chain locus. The endogenous lambda locus is modified by homologousrecombination in ES cells followed by the introduction of geneticallymodified ES cells in mouse embryos to obtain genetically adaptedoffspring.

Two regions of the murine lambda locus that together contain allfunctional lambda V regions are subject to deletion.

The first region targeted for homologous recombination-based deletion isa region that is located 408 bp upstream of the start site of the IGLV2gene segment and ends 215 bp downstream of IGLV3 gene segment, includingthe intergenic sequence stretch between these IGLV gene segments. Thesecond region that is subject to a deletion involves the IGLV1 genesegment consisting of a fragment spanning from 392 bp upstream to 171 bpdownstream of the IGLV1 gene segment. As a consequence of these twodeletion steps, all functional V-lambda genes segments are deleted,rendering the locus functionally inactive (FIGS. 5 and 19A-B).

Construction of the Targeting Vectors

Vectors that received 3-9.6 kb flanking arms on the 3′ and 5′ end fusedto the deletion segment were used for targeted homologous recombinationin an ES cell line. Both arms were obtained by PCR means ensuringmaximum homology. In a first step, the mouse genomic sequenceencompassing the Igl V2-V3 regions were replaced with a PuroR cassetteflanked by F3 sites, which yields a constitutive KO allele afterFlp-mediated removal of selection marker (see FIG. 19A). In a secondstep, the mouse genomic sequence encompassing the Igl V1 region wasreplaced with a Neo cassette in ES cell clones which already carried adeletion of the Igl V2-V3 regions (see FIG. 19B). The selection marker(NeoR) was flanked by FRT sites. A constitutive KO allele was obtainedafter Flp-mediated removal of selection markers.

Generation of ES Cells Bearing the Deleted Lambda Fragment

The generation of genetically modified ES cells was essentiallyperformed as described (J. Seibler, B. Zevnik, B. Küter-Luks, S.Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N. Faust, G.Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kühn, F. Schwenk(2003), Nucleic Acids Res., February 15; 31(4):e12). See also, Example14 for a detailed description. To show that both targeting eventsoccurred on the same chromosome several double targeted clones wereselected for the in vitro deletion with pCMV C31deltaCpG. The cloneswere expanded under antibiotic pressure on a mitotically inactivatedfeeder layer comprised of mouse embryonic fibroblasts in DMEM HighGlucose medium containing 20% FCS (PAN) and 1200 μ/mL LeukemiaInhibitory Factor (Millipore ESG 1107). 1×10⁷ cells from each clone wereelectroporated with 20 μg of circular pCMV C31deltaCpG at 240 V and 500μF and plated on four 10 cm dishes each. Two to three days afterelectroporation, cells were harvested and analyzed by PCR. Primers usedwere:

(SEQ ID NO: 1) 2005_5: CCCTTTCCAATCTTTATGGG (SEQ ID NO: 2)2005_7: AGGTGGATTGGTGTCTTTTTCTC (SEQ ID NO: 3)2005_9: GTCATGTCGGCGACCCTACGCC

PCR reactions were performed in mixtures comprising 5 μl PCR Buffer 10×(Invitrogen), 2 μl MgCl₂ (50 mM), 1 μl dNTPs (10 mM), 1 μl first primer(5 μM), 1 μl second primer (5 μM), 0.4 μl Taq (5 U/ul, Invitrogen), 37.6μl H₂O, and 2 μl DNA. The program used was 95° C. for five minutes;followed by 35 cycles of 95° C. for 30 seconds; 60° C. for 30 seconds;72° C. for 1 minute; followed by 72° C. for ten minutes.

Generation of ES Mice by Tetraploid Embryo Complementation

The production of mice by tetraploid embryo complementation usinggenetically modified ES cells was essentially performed as described(Eggan et al., PNAS 98:6209-6214; J. Seibler, B. Zevnik, B. Küter-Luks,S. Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N. Faust, G.Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kuhn, and F. Schwenk(2003), Nucleic Acids Res., February 15; 31(4):e12; Hogan et al. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y.), pp. 253-289).

Example 5: Construction of the CAGGS Expression Insert Based on aRearranged Human Germline IGKV1-39/J-Ck Gene (IGKV1-39/J-Ck)

This example describes the construction of a CAGGS expression cassetteincorporating the rearranged human germline IGKV1-39/J region. Thisinsert expression cassette encompasses cloning sites, a Kozak sequence,a leader sequence containing an intron, an open reading frame of therearranged IGKV1-39 region, a rat CK constant region from allele a and atranslational stop sequence (IGKV1-39/J-Ck; FIG. 6). The primaryconstruct consists of naturally occurring sequences and has beenanalyzed and optimized by removing undesired cis acting elements likeinternal TATA-boxes, poly adenylation signals, chi-sites, ribosomalentry sites, AT-rich or GC-rich sequence stretches, ARE-, INS- and CRSsequence elements, repeat sequences, RNA secondary structures, (cryptic)splice donor and acceptor sites and splice branch points (GeneArt GmbH).In addition, the codon usage in the open reading frame regions isoptimized for expression in mice. The intron sequence is unchanged andthus represents the sequence identical to the coding part of the humanIGKV1-39 leader intron.

At the 5′ end of the expression cassette, a NotI site was introduced andon the 3′ site a NheI site. Both sites are used for cloning in the CAGGSexpression module. After gene assembly according to methods used byGeneArt, the insert is digested with NotI-NheI and cloned into theexpression module containing a CAGGS promoter, a stopper sequenceflanked by LoxP sites (“floxed”), a polyadenylation signal sequence and,at the 5′ and 3′ end, sequences to facilitate homologous recombinationinto the Rosa26 locus of mouse ES cell lines. Promoter and/or cDNAfragments were amplified by PCR, confirmed by sequencing and/or cloneddirectly from delivered plasmids into an RMCE exchange vector harboringthe indicated features. A schematic drawing and the confirmed sequenceof the final targeting vector pCAGGS-IgVK1-39 are shown in FIGS. 13A and13B. The targeting strategy is depicted in FIG. 13C.

Example 6: CAGGS Expression Insert Based on the Rearranged GermlineIGLV2-14/J V Lambda Region (IGLV2-14/J-Ck)

This example describes the sequence and insertion of an expressioncassette incorporating the rearranged germline IGLV2-14/J V lambdaregion. This insert encompasses cloning sites, a Kozak sequence, aleader sequence containing an intron, an open reading frame of therearranged IGLV2-14/J region, a rat CK constant region from allele a anda translational stop sequence (IGLV2-14/J-Ck; FIG. 7). The primaryconstruct consists of naturally-occurring sequences and has beenanalyzed and optimized by removing undesired cis acting elements like:internal TATA-boxes, poly adenylation signals, chi-sites, ribosomalentry sites, AT-rich or GC-rich sequence stretches, ARE-, INS- and CRSsequence elements, repeat sequences, RNA secondary structures, (cryptic)splice donor and acceptor sites and splice branch points (GeneArt GmbH).In addition, the codon usage in the open reading frame regions wasoptimized for expression in mice. The intron sequence is unchanged andthus represents the sequence identical to the human IGKV1-39 leaderintron.

At the 5′ end of the expression cassette, a NotI site was introduced andon the 3′ site a NheI site. Both sites are used for cloning in the CAGGSexpression module as described by TaconicArtemis. After gene assemblyaccording to methods used by GeneArt, the insert was digested withNotI-NheI and cloned into the expression module containing a CAGGSpromoter, a stopper sequence flanked by LoxP sites (“foxed”), apolyadenylation signal sequence and, at the 5′ and 3′ end, sequences tofacilitate homologous recombination into the Rosa26 locus of mouse EScell lines. To construct the final ROSA26 RMCE targeting vector,promoter and/or cDNA fragments were amplified by PCR. Amplified productswere confirmed by sequencing and/or cloned directly from deliveredplasmids into an RMCE exchange vector harboring the indicated features.A schematic drawing and the confirmed sequence of the final targetingvector pCAGGS-IgVL2-14 is shown in FIGS. 15A and 15B. The targetingstrategy is depicted in FIG. 15C.

Example 7: Expression of IGKV1-39/J-Ck in HEK293 Cell Lines(pSELECT-IGKV1-39/J-Ck)

This example describes a method to verify that the IGKV1-39/J-Ckconstructs described in Example 5 enable expression and detection of theIGKV1-39/J-Ck L chain in HEK293 cells. The IGKV1-39/J insert (FIG. 6)was modified at the 5′ end by changing the NotI site into a SalI site.This change is required for cloning of the product into the expressioncassette plasmid pSELECT-hygro (InvivoGen). The CAGGS expression insertIGKV1-39/J-Ck and pSELECT-hygro were digested with SalI and NheI,ligated and used to transform competent XL1-Blue cells using standardtechniques. Colonies were picked and DNA purified using Qiagen Midi-prepcolumns according to the manufacturer's procedures. The resulting lightchain (LC) expressing vector named 0817676_pSELECT_0815426 was used totransfect HEK293 cells with Fugene6 (Roche) according to themanufacturer's protocols. Supernatants were screened for the presence ofIGKV1-39/J-Ck light chains by ELISA and western blot using anti-rat-Ckantibodies (Beckton Dickinson #550336 and 553871) and protocols used inthe art.

The VH of anti-tetanus toxoid (TT) IgG MG1494 was cloned into IgGexpression vector MV1056 using restriction sites SfiI and BstEII. Theresulting clone was sequence verified. HEK293T cells were transfectedwith five different vector combinations as shown in Table 4 (see Example8 for details of vector 0817678_pSELECT_0815427). Supernatants wereharvested and IgG concentrations determined (see Table 4). No IgG couldbe detected for supernatants A and B containing light chain only asexpected (detection antibody recognized Fc part of IgG). IgGconcentration in supernatants C and D was comparable to that of positivecontrol supernatant E, indicating correct expression of the light chainconstructs.

Binding to TT was analyzed by ELISA to check functionality of theproduced antibodies, using hemoglobin as negative control antigen. NoTT-specific binding could be detected for supernatants A and Bcontaining light chain only, as expected. TT-specific binding forsupernatants C and D was at least as good as for positive controlsupernatant E, confirming correct expression of the light chainconstructs and functional assembly with heavy chain. Antibodies weredetected not only using an anti-human IgG secondary antibody, but alsoan anti-rat Ckappa light chain secondary antibody. The results confirmthat the anti-rat Ckappa antibody (BD Pharmingen #553871, clone MRK-1)recognizes the light chain expressed by the pSELECT vectors.

Supernatants were analyzed by non-reducing SDS-PAGE and Western blot(not shown). Detection using an anti-human IgG heavy chain antibody didnot show bands for supernatants A and B containing light chain only, asexpected. Results for supernatants C and D were comparable to positivecontrol supernatant E, with a band close to the 170 kD marker asexpected for intact IgG. Additional lower molecular weight bands wereobserved as well for supernatants C, D and E, which might representdegradation products, IgG fragments resulting from (partial) reductionand/or irrelevant protein bands due to non-specific binding of thedetection antibody.

Detection using an anti-rat Ckappa light chain antibody showed a bandclose to the 26 kD marker for supernatants A and B, as expected forlight chain only. This band was much more intense for A compared to B,indicating that the free IGKV1-39 light chain may be better expressedand/or more stable than the free IGLV2-14 light chain. No bands weredetected for control supernatant E as expected, since the expressed IgGcontains a human Ckappa light chain. For supernatants C and D, expectedbands close to the 170 kD marker were observed; lower molecular weightbands were also observed, but to a lesser extent than above using theanti-human IgG antibody.

In conclusion, transfection of the light chain expression constructscombined with the heavy chain of anti-tetanus toxoid (TT) IgG MG1494resulted in IgG production comparable to the positive control constructfor both the pSELECT kappa and lambda light chain constructs. Both IgGproductions yielded ELISA signals in a TI′ ELISA that were better thanor comparable to the control IgG. SDS-PAGE and Western blot analysisconfirmed the presence of intact IgG. The tested anti-rat Ckappaantibody worked efficiently in both ELISA and Western blot. Culturesupernatant from cells transfected with light chain constructs only didnot result in detectable IgG production nor in detectable TT-specificbinding, while free light chain was detected on Western blot.

Example 8: Expression of IGLV2-14/J-Ck in HEK293 Cell Lines(pSELECT-IGLV2-14/J-Ck)

This example describes a method to verify that the IGLV2-14/J constructsdescribed in Example 6 enable expression and detection of theIGLV2-14/J-Ck L chain in HEK293 cells. The IGLV2-14/J-Ck insert (FIG. 7)was modified at the 5′ end by changing the NotI site into a SalI site.This change is required for cloning of the product into the expressioncassette plasmid pSELECT-hygro (InvivoGen). The CAGGS expression insertIGLV2-14/J-Ck and pSELECT-hygro were digested with SalI and NheI ligatedand used to transform competentXL1-Blue cells using standard techniques.Colonies were picked and DNA purified using Qiagen Midi-prep columnsaccording to the manufacturer's procedures. The resulting light chain(LC) expressing vector named 0817678_pSELECT_0815427 was used totransfect HEK293 cells with Fugene6 (Roche) according to themanufacturer's protocols. Supernatants were screened for the presence ofIGLV2-14/J-Ck light chains by ELISA and western blot using anti-rat-Ckantibodies (Becton Dickinson #550336 and 553871) and protocols used inthe art. See Example 7 for details and results.

Example 9: Construction of a VK Promoter-Driven Expression ConstructContaining an IGKV1-39/J Insert and Multiple Enhancer Elements Derivedfrom the Murine CK Locus (VkP-IGKV1-39/J-Ck; VkP-O12)

This example describes the construction of an expression cassette thatcontains relevant elements to enable B-cell anddevelopmental/differentiation stage-specific expression of therearranged human IGKV1-39 VK region, based on the IGKV1-39 VK promoterregion, leader containing an intron, germline V-gene, CDR3, IGKJsegment, mouse intergenic region located between Jk and CK, rat Ckallele a open reading frame, and a mouse intergenic fragment from the 3′end of the mouse CK gene ending just 3′ of the 3′ CK enhancer.

Optimized open reading frames of the leader, IGKV1-39 rearranged gene,and rat CK allele a gene, as described in Example 5, was used for theconstruction of the expression cassette. The VK promoter region wasobtained by gene synthesis procedures (GeneArt, GmbH) and is almostidentical to the sequence of the human IGKV1-39 region between −500 bpand the ATG (start site) of the gene. The only deviation from thenatural sequence is the introduction of a GCCACCATGG Kozak sequence (SEQID NO:102) at the ATG (start) site in order to promote translation. Agenomic fragment from a mouse BAC clone (TaconicArtemis) is used as thebasis for the introduction of individual elements. This fragment isidentical to the sequence of the mouse VK locus starting with the introndonor site located directly 3′ of the JK5 region and ending just 3′ ofthe 3′ CK enhancer and covers approximately 12.5 kb.

The final construct contains from 5′ to 3′ end the following elements:human genomic IGKV1-39 promoter (500 bp), a Kozak sequence, a humanIGKV1-39 leader part 1 (optimized), a human IGKV1-39 leader intron, ahuman IGKV1-39 leader part 2 (optimized), a human IGKV1-39 germline gene(optimized), a human J-region (optimized), a mouse intergenic regionincluding the intron enhancer element, a rat (Rattus norvegicus) kappaconstant region (optimized), and a mouse intergenic region including the3′ kappa enhancer. The elements of this expression cassette are shown inFIG. 8 and named VkP-IGKV1-39/J-Ck (VkP-O12). An outline of the pVkP-O12vector and the targeting strategy is depicted in FIGS. 20A and 21A. Thevector was introduced into ES cells following standard procedures (seeExample 14).

Example 10: Construction of a VK Promoter-Driven Expression ConstructContaining an IGLV2-14/J Clone and Multiple CK Locus-Derived EnhancerElements (VkP-IGLVL2-14/J-Ck; VkP-2a2)

This example describes the same construct as described in Example 9,except that the IGKV1-39 gene and J-region are replaced by the optimizedhuman IGLV2-14 germline gene including a unique V-J region(VkP-IGLV2-14/J-Ck; VkP-2a2; FIG. 9).

Example 11: Construction of a VK Promoter-Driven Expression ConstructContaining an IGKV1-39 Clone Lacking the CK Intron Enhancer Element(VkP-IGKV1-39/J-Ck-Δ1; VkP-O12-del1)

The construct described in Example 9 was modified by removing the CKintron enhancer element, located in the intergenic region between thehuman J region and the rat CK region by standard PCR modification andDNA cloning methodologies (GeneArt, GmBH). The resulting expressioncassette is shown in FIG. 10 and named VkP-IGKV1-39/J-Ck-Δ1(VkP-O12-del1).

An outline of the pVkP-O12-del1 vector and the targeting strategy isdepicted in FIGS. 20B and 21B. The vector was introduced into ES cellsfollowing standard procedures (see Example 14).

Example 12: Construction of a VK Promoter-Driven Expression ConstructContaining an IGKV1-39 Clone Lacking the CK Intron Enhancer Element anda Truncated 3′ CK Enhancer Element (VkP-IGKV1-39/J-Ck-Δ2; VkP-O12-del2)

The construct described in Example 11 was modified by truncating the 3′CK enhancer element and deleting part of the intergenic region 3′ of therat Ck gene, to remove potential inhibitory elements. This was achievedby removing the intergenic sequence between an EcoRV site (located 3′ ofthe rat Ck gene) and the NcoI site present in the 3′ enhancer (5993 bp)and further removing the sequence between the 3′ enhancer BstXI site andthe BstXI site 3′ of the 3′ enhancer (474 bp) using standard methods.The resulting expression cassette is shown in FIG. 11 and namedVkP-IGKV1-39/J-Ck-Δ2 (VkP-O12-del2).

An outline of the pVkP-O12-del2 vector and the targeting strategy isdepicted in FIGS. 20C and 21C. The vector was introduced into ES cellsfollowing standard procedures (see Example 14).

Example 13: Expression of Vk Constructs in Cell Lines

The constructs described in Examples 9-12 are tested for their abilityto produce light chain proteins in the myeloma cell lines MPC11 (ATCCCCL167), B-cell lymphoma WEHI231 (ATCC CRL-1702), the T-cell lymphomaEL4 (ATCC TIB-39) and in HEK293 (ATCC CRL1573). The enhancer andpromoter elements in the construct enable expression in the B-cell linesbut not in cell lines derived from other tissues. After transfection ofthe cell lines using purified linearized DNA and Fugene6 (Roche) cellsare cultured for transient expression. Cells and supernatant areharvested and subjected to SDS-PAGE analysis followed by westernblotting using a specific anti-rat-C-kappa antibody. Supernatants areanalyzed in ELISA for secreted L chains using the anti-rat CK antibody(Beckton Dickinson #550336).

Example 14: Generation of Transgenic ES Lines

All constructs as described in Examples 3, 4, 5, 6, 9, 10, 11 and 12were used to generate individual stable transgenic ES lines by means ofhomologous recombination. The methods for generation of transgenic ESlines via homologous recombination are known in the field (e.g., Egganet al., PNAS 98:6209-6214; J. Seibler, B. Zevnik, B. Küter-Luks, S.Andreas, H. Kern, T. Hennek, A. Rode, C. Heimann, N. Faust, G.Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kühn, F. Schwenk(2003), Nucleic Acids Res., February 15; 31(4):e12; Hogan et al. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y.), pp. 253-289).

For all constructs described in Examples 5 and 6, and Examples 9-12, theRMCE ES cell line (derived from mouse strain129S6B6F1-Gt(ROSA)26Sortm10Arte) was grown on a mitotically inactivatedfeeder layer comprised of mouse embryonic fibroblasts (MEF) in DMEM HighGlucose medium containing 15% FBS (PAN 1302-P220821). LeukemiaInhibitory Factor (Chemicon ESG 1107) was added to the medium at aconcentration of 900 U/mL. For manipulation, 2×10⁵ ES-cells were platedon 3.5 cm dishes in 2 ml medium. Directly before transfection, 2 mlfresh medium was added to the cells. Three μl Fugene6 Reagent (Roche;Catalog No. 1 814 443) was mixed with 100 μl serum free medium (OptiMEMI with Glutamax I; Invitrogen; Catalog No. 51985-035) and incubated forfive minutes. One hundred μl of the Fugene/OptiMEM solution was added to2 μg circular vector and 2 μg CAGGS-Flp and incubated for 20 minutes.This transfection complex was added dropwise to the cells and mixed.Fresh medium was added to the cells the following day. From day 2onwards, the medium was replaced daily with medium containing 250 μg/mLG418 (Geneticin; Invitrogen; Catalog No. 10131-019). Seven days aftertransfection, single clones were isolated, expanded, and molecularanalyzed by Southern blotting according to standard procedures.

For each construct, analysis of multiple clones by restriction enzymedigestion of genomic DNA of single clones followed by hybridization with5′ probes, 3′ probes, and internal probes resulted in clones thatcomprised a correct, single insertion at the correct position in theRosa26 locus. An example is provided in FIGS. 14A-C.

Example 15: Generation of Transgenic Mouse Strains

All ES cell lines that were generated and verified for theirmodifications as described in Example 14 were used to generate stabletransgenic mice by means of tetraploid recombination. The methods areknown in the field. In general, after administration of hormones,superovulated Balb/c females were mated with Balb/c males. Blastocystswere isolated from the uterus at dpc 3.5. For microinjection,blastocysts were placed in a drop of DMEM with 15% FCS under mineraloil. A flat tip, piezo actuated microinjection-pipette with an internaldiameter of 12-15 micrometers was used to inject 10-15 targeted C57BL/6N.tac ES cells into each blastocyst. After recovery, injectedblastocysts were transferred to each uterine horn of 2.5 days postcoitum, pseudopregnant NMRI females. Chimerism was measured in chimeras(G0) by coat color contribution of ES cells to the Balb/c host(black/white). Highly chimeric mice were bred to strain C57BL/6 females.Depending on the project requirements, the C57BL/6 mating partners arenon-mutant (W) or mutant for the presence of a recombinase gene(Flp-Deleter or Cre-deleter or CreER inducible deleter or combination ofFlp-deleter/CreER). Germline transmission was identified by the presenceof black, strain C57BL/6, offspring (G1).

For example, ESC clone IgVK1-39 2683 8 (see Examples 5 and 14) wasinjected in a total of 62 blastocysts in three independent experiments.Three litters were obtained with a total of six pups. All pups werechimeric. Three heterozygous offspring pups were obtained that were usedfor further crossing.

ESC Clone Kappa 2692 A-C10 (see Examples 3 and 14) was injected in atotal of 54 blastocysts in three independent experiments. Three litterswere obtained with a total of eleven pups, of which ten were chimeric.Eight heterozygous offspring pups were obtained that were used forfurther crossing.

ESC Clone Kappa 2692 B-C1 (see Examples 3 and 14) was injected in atotal of 51 blastocysts in three independent experiments. Two litterswere obtained with a total of six pups, of which four were chimeric.Three heterozygous offspring pups were obtained that were used forfurther crossing.

Example 16: Breeding

This example describes the breeding for obtaining mice that containtransgenic expression cassettes as described Example 14 and knock-outmice in which the endogenous lambda and kappa loci have been silenced.The localization of V-lambda on chromosome 16 and CD19 on chromosome 7allow standard breeding procedures. The breeding of the co-localized Vklocus and Rosa26 locus on chromosome 6 with a distance of about 24 cMrequires special attention during the screening as only a percentage ofthe offspring shows crossover in a way that both modifications arebrought together on one chromosome.

All four loci have to be combined in a single mouse strain that is homo-or heterozygous for CD19-cre (not described) and modified Rosa26transgene and homozygous for the other loci. Breeding is performed bystandard breeding and screening techniques as appropriate and offered bycommercial breeding companies (e.g., TaconicArtemis).

Example 17: Immunizations of Mice

Primary and booster immunization of mice are performed using standardprotocols.

To validate the transgenic expression of human rearranged Vκ O12(IGKV1-39)-rat C_(κ) light chains (see Examples 5, 14-16) in B cellsfrom CD19-HuVκ1 mice and to assess its impact on VH repertoire size,diversity of VH family usage and V(D)J recombination after immunization,the CD19-HuVκ1 transgenic mice are immunized with tetanus toxin vaccine(TI′ vaccine) and VH sequence diversity of randomly picked clones fromCD19-HuVκ1 mice are compared with TT-immunized wt mice and CD19-CreHuVk1 negative littermates. Data on the SHM frequency of the human VκO12 transgene in the immunized mice are obtained. A diverse collectionof at least 40 TT-specific, clonally-unrelated mAbs containing the humanVκ O12 are recovered from CD19-HuVκ1 mice by phage display.

For this, three adult CD19-HuVκ1 mice are vaccinated with TT vaccineusing standard immunization procedures. After immunization, serum titersare measured using TT specific ELISA (TT: Statens Serum Institute, Art.no. 2674) and spleen suspensions subjected to cell sorting by the FACSprocedure after staining with a rat Cκ-specific monoclonal antibody toisolate transgenic B cells (clone RG7/9.1; BD Pharmingen#553901,Lot#06548). RNA from rat Cκ-positive B cells are extracted and theresulting cDNA material used for library building and SHM analysis.

The standard monoclonal mouse anti-rat CK antibody (clone RG7/9.1; BDPharmingen#553901, Lot#06548) is used in FACS analysis of transgeneexpressing B cells (Meyer et al. (1996), Int. Immunol. 8:1561). Theclone RG7/9.1 antibody reacts with a monotypic (common) kappa chaindeterminant. This anti-rat Cκ antibody (clone RG7/9.1 (BDPharmingen#553901, Lot#06548) is labeled with R-phycoerythrin (PE) usingthe LYNX rapid conjugation kit according to the manufacturer'sinstructions for FACS analysis and sorting. The labeled antibody isfirstly tested by flow cytometry for binding to rat CK-containingfunctional light chain proteins produced into transiently transfectedHEK-293T cells; the un-conjugated antibody serves as a positive control.Two other antibodies shown to bind to rat Cκ by ELISA and Western-blot(see Example 7) are tested as well by flow cytometry.

Fab-phage display library building is carried out with a set ofoptimized degenerate PCR primers designed to amplify C57BL/6 VH genes;the minimal library size is 10⁶ clones, and minimal insert frequency is80%. The vector used, MV1043 (FIGS. 3 and 12), contains the human Vκ O12fused to a human Cκ region. The rat Cκ is therefore exchanged for thehuman counterpart in the library generation process.

Before selection, VH sequencing of 96 randomly picked clones isperformed to validate VH repertoire diversity that is compared todiversity obtained from an unselected library previously generated usingthe same procedures from BALB/c mice immunized with TT. A library fromC57Bl/6 wt mice that are immunized in the same way allows diversitycomparison between two preselected libraries sharing the same vaccineand the same genetic background.

Several independent selections are performed on TT coated inimmunotubes. Variables that may be included are selections usingbiotinylated antigens in solution or selections on captured TT. Based onthe number and diversity of ELISA-positive clones obtained in the firstselections, decisions on additional rounds of selection are made. Clonesare considered positive when >3× positive over a negative control clone.Positive clones are analyzed by ELISA against a panel of negativecontrol antigens to verify antigen specificity. The aim is to identifyat least 40 unique VH regions, as based on unique CDR3 sequences andV_(H)DJ_(H) rearrangements.

Amplification of the cDNA material from rat CK-positive sorted B cellsis performed with a PCR forward primer specific to the human leadersequence and a PCR reverse primer specific to the rat CK sequence, in aregion not redundant with the mouse CK sequence, as reported in a recentstudy (Brady et al. (2006), JIM 315:61). Primer combinations andannealing temperatures are firstly tested on cDNA from HEK-293T cellstransfected with 0817676_pSELECT_0815426=pSELECT vector with IGKV1-39DNA cassette (see Example 7).

The amplification products is cloned in pJET-1 vector and after XL1-bluetransformation, 96 colonies are sequenced for assessing VL SHM frequencyby direct comparison to the Vκ O12 (IGKV1-39) germline sequence. The R/Sratio method, as described in our study on human IT-specific antibodies(de Kruif et al. (2009), J. Mol. Biol. 387:548) allows discriminationbetween random mutations and antigen-driven mutations that occurred onVL sequences.

Example 18: Immunofluorescent Analysis of B Cell Populations inTransgenic Mouse Lines

This example describes the use of antibodies and flow cytometry toanalyze B cell populations in primary (bone marrow) and secondary(spleen, peritoneal) lymphoid organs and blood. Methods and reagents aredescribed in Middendorp et al. (2002), J. Immunol. 168:2695; andMiddendorp et al. (2004), J. Immunol. 172:1371. For analysis of early Bcell development in bone marrow, cells were surface stained withcombinations of antibodies (Becton Dickinson) specific for B220, CD19,CD25, IgM, IgD, mouse Ckappa, mouse Clambda and rat Ckappa to detectpro-B cells, pre-B cells, large pre-B cells, early and late immature Bcells and recirculating B cell populations expressing the transgene ontheir surface. DAPI staining (Invitrogen) was included to exclude deadcells from the analysis and FC block (Becton Dickinson) to inhibitantibody interaction with Fc receptors on myeloid cells. For analysis ofsurface transgene expression on B cell populations in peripherallymphoid organs and blood, cells were stained with combinations ofantibodies (Becton Dickinson) specific for B220, CD5, CD19, CD21, CD23,IgM, IgD, mouse Ckappa, mouse Clambda and rat Ckappa. DAPI staining wasincluded to exclude dead cells from the analysis and FC block to inhibitantibody interaction with Fc receptors on myeloid cells. In addition,combinations of antibodies (Becton Dickinson) specific for CD3, CD4,CD11b, CD11c and NK1.1 were included to determine if transgeneexpression occurred in cell types outside of the B cell compartment.

Three mice heterozygous for the human IGKV1-39/rat Ckappa transgene andheterozygous for the CD19-Cre transgene on a C57BL6 background(HuVk1/CD19-Cre) were analyzed. As controls for the FACS analysis, threelittermate mice wild-type for the human IGKV1-39/rat Ckappa transgeneand heterozygous for the CD19-Cre transgene on a C57BL6 background(CD19-Cre) and two C57BL6/NTac mice (Wt) were included. All animals wereallowed to acclimatize in the animal facility for one week beforeanalysis and all mice were male and six weeks of age. Lymphocytes wereisolated from the femurs, spleens, peritoneal cavity and blood of miceusing conventional techniques as previously described (Middendorp et al.(2002), J. Immunol. 168:2695; and Middendorp et al. (2004), J. Immunol.172:1371). Antibodies were pre-combined as shown in FIG. 29A-B andstaining was carried out in 96-well plates. Incubation with thePE-conjugated anti-rat C kappa (described above) was carried out beforestaining with the rat anti-murine antibodies to avoid non-specificbinding. After completion of cell staining, labeled cells were analyzedon a Becton Dickinson LSR II FACS machine and the acquired data analyzedwith FlowJo software (v6.4.7).

Transgenic mice were similar in weight, appearance and activity towild-type mice. No gross anatomical alterations were observed during theharvesting of tissues. No difference was observed in the numbers of Bcells in the bone marrow (BM) and spleen (Table 9) or in the numbers ofB cells, T cells and myeloid cells in peripheral organs betweentransgenic and wild-type mice. In addition, the frequency or proportionof the cells in the different lymphocyte developmental pathways was notaltered in transgenic mice when compared to wild-type mice. Thus in thedouble transgenic (HuVk1/CD19-Cre) and transgenic (CD19-Cre) micelymphoid and most importantly B cell development was indistinguishablefrom wild-type mice.

In the peripheral lymphoid organs, staining with the transgene specificantibody (anti-ratCkappa-PE) was only observed in the B cellpopulations. T cell, myeloid cell and NK cell populations were allnegative for surface expression of the transgene in the spleen (FIG.23). In contrast, in cells stained with the pan B cell markers B220 andCD19 all cells were shifted to the right in the FACS plot indicatingcell surface expression of the transgene (FIG. 24). A similartransgene-specific staining was measured in CD5⁺ B1 cells of theperitoneum, a developmentally distinct population of B cells (FIG. 25).

Differentiation of B cells from multilineage precursors to mature Bcells occurs in the bone marrow. In the lymphocytes analyzed from thebone marrow, extracellular and transgene expression was not detectablein the earliest B cell progenitors the pro- and pre-B cell consistentwith the pattern of normal light chain expression (FIGS. 26A-B).Transgene expression first becomes detectable in immature B cells, thedevelopmental stage at which the germline murine light chain undergoesrearrangement and is expressed at the cell surface in the context of thepreselected heavy chain (FIGS. 26A-B). Consistent with the staining inthe spleen transgenic light chain expression is also detected on maturerecirculating B cells (FIGS. 26A-B). Thus the CD19-Cre driven expressionof the transgene is consistent with the normal pattern of light chainexpression. The staining with the endogenous light chain-specificantibody is more intense than that of the transgene-specific light chainantibody. This may indicate a higher expression level of the endogenouslight chain, a more sensitive staining with the endogenous lightchain-specific antibody or a combination of both. Importantly, theintensity of the surface expression of the transgenic light chain iscorrelated with both endogenous light chain and IgM surface expressionas observed in staining of circulating B cells in the blood (FIG. 27).

Thus, overall this analysis demonstrates that expression of the humanIGKV1-39/Ckappa transgene is restricted to the B cell compartment andthe temporal regulation of its expression is similar to the endogenouskappa and lambda light chains resulting in normal development of all Bcell populations. The apparent lower level of expression of thetransgene could be explained by the strength of the promoter incomparison to the promoter and enhancers present on endogenous lightchain genes or by a delay in transgene expression that gives theendogenous light chains a competitive advantage in pairing with therearranged heavy chain. This is consistent with the observation that asB cells mature the relative intensity of transgene staining increasescompared to the endogenous light chains. In addition, the observationthat B cells numbers are normal and that every surface Ig+ B cellco-expresses an endogenous and transgenic light chain supports theconclusion that the IGKV1-39 variable region is capable of pairing witha normal repertoire of different murine heavy chain variable regions. Weconclude from this analysis that insertion of the IGKV1-39/rat Ckappatransgene driven by the CD19-Cre activated CAGGS promoter in the Rosalocus facilitates timely and B cell-specific expression of the transgeneand that the transgene is capable of pairing with a normal repertoire ofmurine heavy chains.

Example 19: Epibase® T-Cell Epitope Profile for IGKV1-39

The protein sequence of IGKV1-39 (FIG. 12, human germline IGKV1-39/JProtein) was scanned for the presence of putative HLA class IIrestricted epitopes, also known as T_(H)-epitopes. For this, Algonomics'Epibase® platform was applied to IGKV1-39. In short, the platformanalyzes the HLA binding specificities of all possible 10-mer peptidesderived from a target sequence (Desmet et al. (1992), Nature356:539-542; Desmet et al. (1997), FASEB J. 11:164-172; Desmet et al.(2002), Proteins 48:31-43; Desmet et al. (2005), Proteins 58:53-69).Profiling is done at the allotype level for 20 DRB1, 7 DRB3/4/5, 13 DQand 7 DP, i.e., 47 HLA class II receptors in total (see Table 5).Epibase® calculates a quantitative estimate of the free energy ofbinding ΔG_(bind) of a peptide for each of the 47 HLA class IIreceptors. These data were then further processed as follows:

Free energies were converted into Kd-values through ΔG_(bind)=RT ln(Kd).

Peptides were classified as strong (S), medium (M), weak and non (N)binders. The following cutoffs were applied:

S: strong binder: Kd<0.1 μM.

M: medium binder: 0.1 μM≦Kd<0.8 μM.

N: weak and non-binder: 0.8 μM≦Kd.

Peptides corresponding to self-peptides were treated separately. Thelist of self-peptides was taken from 293 antibody germline sequences.They are referred to as “germline-filtered” peptides.

S- and M-peptides are mapped onto the target sequence in so-calledepitope maps; S-affinities are plotted quantitatively; M-values arepresented qualitatively. As a general overview of the results, Table 6lists the number of strong and medium binders in the analyzed proteins,for the groups of HLA class II receptors corresponding to the DRB1, DQ,DP and DRB3/4/5 genes. Counting was done separately for strong andmedium affinity binders. Peptides binding to multiple allotypes of thesame group were counted as one. Values between brackets refer togermline-filtered peptides. In Table 7, the sequence is shown in aformat suitable for experimental work. The sequence is broken down inconsecutive 15-mers overlapping by 12 residues. For each 15-mer, thepromiscuity is listed (the number of allotypes out of a total of 47 forwhich the 15-mer contains a critical binder), as well as the impliedserotypes. The Epibase® profile and epitope maps are shown in FIGS.16A-C and 17.

It was concluded that IGKV1-39 contains no strong non-self DRB1 binders.Typically, significantly more binders were found for DRB1 than for otherHLA genes. This is in agreement with experimental evidence thatallotypes belonging to the DRB1 group are more potent peptide binders.Medium strength epitopes for DRB1 allotypes are expected to contributeto the population response, and cannot be disregarded. Again, nonon-self DRB1 binders were found in IGKV1-39.

In the humoral response raised against an antigen, the observed T_(H)cell activation/proliferation is generally interpreted in terms of theDRB1 specificity. However, one cannot ignore the possible contributionof the DRB3/4/5, DQ and DP genes. Given the lower expression levels ofthese genes as compared to DRB1, the focus was on the class of strongepitopes for DRB3/4/5, DQ and DP. “Critical epitopes” are those epitopesthat are strong binders for any DRB1, DRB3/4/5, DQ or DP allotype or aremedium binders for DRB1. IGKV1-39 contains no strong or medium non-selfbinders for DRB3/4/5, DQ, or DP.

A number of peptides are also present in germline sequences (valuesbetween brackets in Table 6). Such peptides may very well bind to HLAbut they are assumed to be self and, hence, non-immunogenic. In total,six strong and 16 medium germline-filtered DRB1 binders were found inIGKV1-39. Framework region 1 up to framework region 3 is an exact matchfor germline V-segment VKI 2-1-(1) O12 (VBase), a.k.a. IGKV1-39*01(IMGT). Framework region 4 is an exact match for germline J-segment JK1(V-base) a.k.a. IGKJ1*01(IMGT). It is hardly surprising that thesesegments do not contain any non-self epitopes.

Example 20: Production Characteristics of IGKV1-39

There is a great demand for antibody discovery platforms that yieldtherapeutic antibodies that are thermodynamically stable and give goodexpression yields. These characteristics are important in ensuring thestability of the drug substance during production and after injection ofthe drug product into the patient. In addition good expression yieldsimpact directly on the cost of drug manufacture and thus pricing,patient access and profitability. Virtually all therapeutic antibodiesin clinical use today are composed of human IgG1 and kappa constantregions but use different heavy and light chain variable regions thatconfer specificity. Human variable heavy and light chain domains can bedivided into families that have greater than 80% sequence divergence.When rearranged examples of these families in germline configuration arecombined and compared for stability and yield it is clear that the genefamilies are not equal in terms of biophysical properties. In particularV_(H)3, V_(H)1 and V_(H)5 have favourable stability for the heavy chainsand Vk1 and Vk3 have the best stability and yield of light chains. Inaddition when mutations are introduced as part of the somatichypermutation process they can interfere with V_(H)/V_(L) pairing. Toassess the effect that different light chain genes with different ratesof mutation have on the production characteristics of a fixed V_(H)chain, a Fab phage display library was built of light chains (kappa andlambda) from six naïve healthy donors combined with a panel of 44 TTbinding heavy chains from immunized donors. After one round of selectionTT binding Fab clones were isolated. Several of these shared the sameV_(H) gene as the TT clone PG1433 in combination with different lightchains. The Fab light chain fragments were recloned into a kappaexpression vector and transfected in combination with DNA encoding theheavy chain of PG1433 into 293 cells and specific IgG productionmeasured by ELISA. As demonstrated in Table 8 the selected clonescontaining PG1433 V_(H) combined with different light chains had betweenfive- and ten-fold lower protein expression PG1433 V_(H) combined withIGKV1-39. Note that all of the light chains contained amino acidmutations within their coding regions that might disrupt V_(H) paringand reduce production stability. Thus, in addition to reducing thechances of unwanted immunogenicity, it is expected that the use of thelight chain IGKV1-39 without mutations contributes to improvedproduction stability and yields of various specificity-contributingV_(H) genes. Indeed stable clones generated by the transfection ofdifferent V_(H) genes all paired with IGKV1-39 are able to be passagedextensively and still retain robust production characteristics as shownin FIG. 28.

Example 21: Generation of Mice Expressing Fully Human VH and VL Regions

Transgenic mice described herein are crossed with mice that alreadycontain a human VH locus. Examples of appropriate mice comprising ahuman VH locus are disclosed in Taylor et al. (1992), Nucleic Acids Res.20:6287-95; Lonberg et al. (1994), Nature 368:856-9; Green et al.(1994), Nat. Genet. 7:13-21; Dechiara et al. (2009), Methods Mol. Biol.530:311-24.).

After crossing and selecting for mice that are at least heterozygous forthe IGKV1-39 transgene and the human VH locus, selected mice areimmunized with a target. VH genes are harvested as describedhereinabove. This method has the advantage that the VH genes are alreadyfully human and thus do not require humanization.

Example 22: Isolation, Characterization, Oligoclonics Formatting andProduction of Antibodies Targeting Human IL6 for Treatment of ChronicInflammatory Diseases Such as Rheumatoid Arthritis

A spleen VH repertoire from transgenic mice that are immunized withhuman recombinant IL6 is cloned in a phage display Fab vector with asingle human IGKV1-39-C kappa light chain (identical to the mousetransgene) and subjected to panning against the immunogen human IL6.Clones that are obtained after two to four rounds of panning areanalyzed for their binding specificity. VH genes encoding IL6-specificFab fragments are subjected to sequence analysis to identify uniqueclones and assign VH, DH and JH utilization. The Fab fragments arereformatted as IgG1 molecules and transiently expressed. Unique clonesare then grouped based on non-competition in binding assays andsubjected to affinity and functional analysis. The most potent anti-IL6IgG1 mAbs are subsequently expressed as combinations of two, three, fouror five heavy chains comprising different VH-regions in the Oligoclonicsformat, together with one IGKV1-39-C-based kappa light chain and testedin vitro for complex formation with IL-6. The Oligoclonics are alsotested in vivo for clearance of human IL-6 from mice. An Oligoclonicwith the most potent clearance activity is chosen and the murine VHgenes humanized according to conventional methods. The humanized IgG1are transfected into a mammalian cell line to generate a stable clone.An optimal subclone is selected for the generation of a master cell bankand the generation of clinical trial material.

Many of the protocols described here are standard protocols for theconstruction of phage display libraries and the panning of phages forbinding to an antigen of interest and are described, for example, inAntibody Phage Display: Methods and Protocols (2002), Editor(s) PhilippaM. O'Brien, Robert Aitken, Humana Press, Totowa, N.J., USA.

Immunizations

Transgenic mice receive three immunizations with human IL6 every twoweeks using the adjuvant Sigma titerMax according to manufacturer'sinstructions.

RNA Isolation and cDNA Synthesis

Three days after the last immunization, spleens and lymphnodes from themice are removed and passed through a 70 micron filter into a tubecontaining PBS pH 7.4 to generate a single cell suspension. Afterwashing and pelleting of lymphocytes, cells are suspended in TRIzol LSReagent (Invitrogen) for the isolation of total RNA according to themanufacturer's protocol and subjected to reverse transcription reactionusing 1 microgram of RNA, Superscript III RT in combination with dT20according to manufacturer's procedures (Invitrogen).

The generation of Fab phage display libraries is carried out asdescribed in Example 2.

Selection of Phages on Coated Immunotubes

Human recombinant IL6 is dissolved in PBS in a concentration of 5 μg/mland coated to MAXISORP™ Nunc-Immuno Tube (Nunc 444474) overnight at 4°C. After discarding the coating solution, the tubes are blocked with 2%skim milk (ELK) in PBS (blocking buffer) for one hour at RoomTemperature (RT). In parallel, 0.5 ml of the phage library is mixed with1 ml blocking buffer and incubated for 20 minutes at room temperature.After blocking the phages, the phage solution is added to the IL6-coatedtubes and incubated for two hours at RT on a slowly rotating platform toallow binding. Next, the tubes are washed ten times with PBS/0.05%TWEEN™-20 detergent followed by phage elution by incubating with 1 ml 50mM glycine-HCl pH 2.2 ten minutes at RT on rotating wheel and directlyfollowed by neutralization of the harvested eluent with 0.5 ml 1 MTris-HCl pH 7.5.

Harvesting Phage Clones

A 5 ml XL1-Blue MRF (Stratagene) culture at O.D. 0.4 is added to theharvested phage solution and incubated for 30 minutes at 37° C. withoutshaking to allow infection of the phages. Bacteria are plated onCarbenicillin/Tetracycline 4% glucose 2*TY plates and grown overnight at37° C.

Phage Production

Phages are grown and processed as described by Kramer et al. 2003(Kramer et al. 2003, Nucleic Acids Res. 31(11):e59) using VCSM13 ashelper phage strain.

Phage ELISA

ELISA plates are coated with 100 microliters human recombinant IL6 perwell at a concentration of 2.5 micrograms/ml in PBS overnight at 4° C.Plates coated with 100 microliters thyroglobulin at a concentration of 2micrograms/ml in PBS are used as a negative control. Wells are emptied,dried by tapping on a paper towel, filled completely with PBS-4% skimmedmilk (ELK) and incubated for one hour at room temperature to block thewells. After discarding the block solution, phage minipreps pre-mixedwith 50 μl blocking solution are added and incubated for one hour at RT.Unbound phages are subsequently removed by five washing steps withPBS-0.05% Tween-20. Bound phages are detected by incubating the wellswith 100 microliters anti-M13-HRP antibody conjugate (diluted 1/5000 inblocking buffer) for one hour at room temperature. Free antibody isremoved by repeating the washing steps as described above, followed byTMB substrate incubation until color development was visible. Thereaction is stopped by adding 100 microliters of 2 M H2SO4 per well andanalyzed on an ELISA reader at 450 nm emission wavelength.

Sequencing

Clones that give signals at least three times above the backgroundsignal are propagated, used for DNA miniprep procedures (see proceduresQiagen miniPrep manual) and subjected to nucleotide sequence analysis.Sequencing is performed according to the Big Dye 1.1 kit accompanyingmanual (Applied Biosystems) using a reverse primer (CH1_Rev1, Table 1)recognizing a 5′ sequence of the CH1 region of the human IgG1 heavychain (present in the Fab display vector MV1043, FIGS. 3 and 12). Thesequences of the murine VH regions are analyzed for diversity of DH andJH gene segments.

Construction and Expression of Chimeric IgG1

Vector MV1057 (FIGS. 12 and 22) was generated by cloning the transgene(IGKV1-39) L chain fragment into a derivative of vector pcDNA3000Neo(Crucell, Leiden, The Netherlands) that contains the human IgG1- andkappa constant regions. VH regions are cloned into MV1057 and nucleotidesequences for all constructs are verified according to standardtechniques. The resulting constructs are transiently expressed inHEK293T cells and supernatants containing chimeric IgG1 are obtained andpurified using standard procedures as described before (M. Throsby 2006,J. Virol. 80:6982-92).

IgG1 Binding and Competition Analysis

IgG1 antibodies are titrated in ELISA using IL6-coated plates asdescribed above and an anti-human IgG peroxidase conjugate. CompetitionELISAs to group antibodies based on epitope recognition are performed byincubating Fab phages together with IgG1 or with commercial antibodiesagainst IL6 (e.g., Abcam cat. no. ab9324) in IL6-coated plates, followedby detection of bound Fab phage using an anti-M13 peroxidase conjugate.

IgG1 Affinity Measurements

The affinities of the antibodies to IL6 are determined with theQuantitative kinetic protocol on the Octet (ForteBio). Antibodies arecaptured onto an Anti-Human IgG Fc Capture biosensor and exposed to freeIL6 and analyzed using proprietary software to calculate the Kd of eachantibody.

Functional Activity of IL6 Antibodies

To test the ability of the selected antibodies to inhibit bindingbetween IL6 and IL6 receptor (IL6R), an ELISA based assay is used.Various concentrations of antibody are mixed with a fixed concentration(10 ng/ml) of biotinylated IL6 as described by Naoko et al. 2007, Can.Res. 67:817-875. The IL6-antibody immune complex is added to immobilizedIL6R. The binding of biotinylated IL6 to IL6R is detected withhorseradish peroxidase-conjugated streptavidin. The reduction of ELISAsignal is a measurement of inhibition. As positive control forinhibition of binding between IL6 and IL6R either anti-IL6R antibody(Abcam cat. no. ab34351; clone B-R6) or anti IL6 antibody (Abcam cat.no. ab9324) is used.

In vitro blocking activity of the selected anti-IL6 antibodies ismeasured in a proliferation assay using the IL6-dependent cell line7TD1. Briefly, cells are incubated with different concentrations ofhuman IL6 with or without the anti-IL6 antibody. The available amount ofIL6 determines the degree of proliferation. Thus if an added antibodyblocks IL6 binding the proliferation readout is reduced compared to anon binding antibody control. Proliferation is measured by theincorporation of 5-bromo-2′-deoxy-uridine (BrdU) into the DNA using theBrdU proliferation kit (Roche cat. no. 11444611001) according to themanufacturer's instructions.

Generation of Anti-IL6 Oligoclonics

The most potent anti-IL6 antibodies are selected from each epitopegroup. The expression constructs expressing these antibodies aretransfected into HEK293T cells in non-competing groups of three indifferent ratios (1:1:1; 3:1:1; 1:3:1; 1:1:3; 3:3:1; 1:3:3; 3:1:3;10:1:1; 1:10:1; 1:1:10; 10:10:1; 1:10:10; 10:1:10; 3:10:1; 10:3:1;1:10:3; 3:1:10; 10:1:3; 1:3:10). Antibody containing supernatants areharvested and purified and characterized as above.

Complex Formation and In Vivo Clearance of Anti-IL6 Oligoclonics

To measure the ability of anti-IL6 Oligoclonics to form immune complexesand to analyze these complexes Size Exclusion Chromatography (SEC) isused according to the approach disclosed by Min-Soo Kim et al. (2007),JMB 374:1374-1388, to characterize the immune-complexes formed withdifferent antibodies to TNFα. Different molar ratios of the anti-IL6Oligoclonics are mixed with human IL6 and incubated for 20 hours at 4°C. or 25° C. The mixture is analyzed on an HPLC system fitted with asize exclusion column; different elution times are correlated tomolecular weight using a molecular weight standards.

The ability of antibodies to form complexes with IL6 is correlated withtheir ability to rapidly clear the cytokine from the circulation invivo. This is confirmed by measuring the clearance of radiolabelled IL6from mice. Briefly, female, six- to eight-week-old Balb/c mice areobtained and 18 hours before the experiment, the animals are injectedintravenously (IV) via the lateral tail vein with different doses ofpurified anti-IL6 Oligoclonics. On day 0, the mice are injected IV with50 microliters of radiolabeled IL-6 (1×10E7 cpm/mL) under the sameconditions. Blood samples (approximately 50 microliters) are collectedat several time intervals and stored at 4° C. The samples arecentrifuged for five minutes at 4000×g and the radioactivity of theserum determined. All pharmacokinetic experiments are performedsimultaneously with three animals for each treatment.

Generation of Anti-IL6 Oligoclonics Stable Clones and PreclinicalDevelopment

A lead anti-IL6 Oligoclonic is selected based on the in vitro and invivo potency as determined above. The murine VH genes are humanizedaccording to standard methods and combined with the fully human IGKV1-39light chain in an expression vector as described above. Examples ofhumanization methods include those based on paradigms such asresurfacing (E. A. Padlan et al. (1991), Mol. Immunol. 28:489),superhumanization (P. Tan, D. A., et al. (2002), J. Immunol. 169:1119)and human string content optimization (G. A. Lazar et al. (2007), Mol.Immunol. 44:1986). The three constructs are transfected into PER.C6cells at the predetermined optimal ratio (described above) under theselective pressure of G418 according to standard methods. A stable highproducing anti-IL6 Oligoclonic clone is selected and a working andqualified master cell bank generated.

TABLE 1 List of primers DO- Primer Sequence 0012 CH1_Rev1TGCCAGGGGGAAGACCGATG (SEQ ID NO: 4) 0656 MVH-1GCCGGCCATGGCCGAGGTRMAGCTTCAGG AGTCAGGAC (SEQ ID NO: 5) 0657 MVH-2GCCGGCCATGGCCGAGGTSCAGCTKCAGC AGTCAGGAC (SEQ ID NO: 6) 0658 MVH-3GCCGGCCATGGCCCAGGTGCAGCTGAAGS ASTCAGG (SEQ ID NO: 7) 0659 MVH-4GCCGGCCATGGCCGAGGTGCAGCTTCAGG AGTCSGGAC (SEQ ID NO: 8) 0660 MVH-5GCCGGCCATGGCCGARGTCCAGCTGCAAC AGTCYGGAC (SEQ ID NO: 9) 0661 MVH-6GCCGGCCATGGCCCAGGTCCAGCTKCAGC AATCTGG (SEQ ID NO: 10) 0662 MVH-7GCCGGCCATGGCCCAGSTBCAGCTGCAGC AGTCTGG (SEQ ID NO: 11) 0663 MVH-8GCCGGCCATGGCCCAGGTYCAGCTGCAGC AGTCTGGRC (SEQ ID NO: 12) 0664 MVH-9GCCGGCCATGGCCCAGGTYCAGCTYCAGC AGTCTGG (SEQ ID NO: 13) 0665 MVH-10GCCGGCCATGGCCGAGGTCCARCTGCAAC AATCTGGACC (SEQ ID NO: 14) 0666 MVH-11GCCGGCCATGGCCCAGGTCCACGTGAAGC AGTCTGGG (SEQ ID NO: 15) 0667 MVH-12GCCGGCCATGGCCGAGGTGAASSTGGTGG AATCTG (SEQ ID NO: 16) 0668 MVH-13GCCGGCCATGGCCGAVGTGAAGYTGGTGG AGTCTG (SEQ ID NO: 17) 0669 MVH-14GCCGGCCATGGCCGAGGTGCAGSKGGTGG AGTCTGGGG (SEQ ID NO: 18) 0670 MVH-15GCCGGCCATGGCCGAKGTGCAMCTGGTGG AGTCTGGG (SEQ ID NO: 19) 0671 MVH-16GCCGGCCATGGCCGAGGTGAAGCTGATGG ARTCTGG (SEQ ID NO: 20) 0672 MVH-17GCCGGCCATGGCCGAGGTGCARCTTGTTG AGTCTGGTG (SEQ ID NO: 21) 0673 MVH-18GCCGGCCATGGCCGARGTRAAGCTTCTCG AGTCTGGA (SEQ ID NO: 22) 0674 MVH-19GCCGGCCATGGCCGAAGTGAARSTTGAGG AGTCTGG (SEQ ID NO: 23) 0675 MVH-20GCCGGCCATGGCCGAAGTGATGCTGGTGG AGTCTGGG (SEQ ID NO: 24) 0676 MVH-21GCCGGCCATGGCCCAGGTTACTCTRAAAG WGTSTGGCC (SEQ ID NO: 25) 0677 MVH-22GCCGGCCATGGCCCAGGTCCAACTVCAGC ARCCTGG (SEQ ID NO: 26) 0678 MVH-23GCCGGCCATGGCCCAGGTYCARCTGCAGC AGTCTG (SEQ ID NO: 27) 0679 MVH-24GCCGGCCATGGCCGATGTGAACTTGGAAG TGTCTGG (SEQ ID NO: 28) 0680 MVH-25GCCGGCCATGGCCGAGGTGAAGGTCATCG AGTCTGG (SEQ ID NO: 29) 0681 ExtMVH-1CAGTCACAGATCCTCGCGAATTGGCCCA

ATGGCCSANG (SEQ ID NO: 30) 0682 ExtMVH-2 CAGTCACAGATCCTCGCGAA TTGGCCCA

ATGGCCSANC (SEQ ID NO: 31) 0683 MJH-Rev1 GGGGGTGTCGTTTTGGCTGAGGAGAC

GTGG (SEQ ID NO: 32) 0684 MJH-Rev2 GGGGGTGTCGTTTTGGCTGAGGAGAC

GTGG (SEQ ID NO: 33) 0685 MJH-Rev3 GGGGGTGTCGTTTTGGCTGCAGAGAC

AGAG (SEQ ID NO: 34) 0686 MJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC

GAGG (SEQ ID NO: 35) 0687 ExtMJH-Rev1& GGGGGTGTCGTTTTGGCTGAGGAGAC

GTGG (SEQ ID NO: 36) 0688 ExtMJH-Rev2in GGGGGTGTCGTTTTGGCTGAGGAGAC

GTGG (SEQ ID NO: 37) 0690 ExtMJH-Rev3 GGGGGTGTCGTTTTGGCTGAGGGAGAC

AGA (SEQ ID NO: 38) 0691 ExtMJH-Rev4 GGGGGTGTCGTTTTGGCTGAGGAGAC

GAGG (SEQ ID NO: 39)

TABLE 2 Phage ELISA signal levels as measured 450 at nm. TT-coatedplates represent plates that were coaret with tetanus toxoid.Thyroglobulin- coated plates are used as negative controls. 10/10 and15/15 indicate the number of wash steps with PBS-Tween during panningprocedures. The 10/10 tetanus toxoid and 10/10 thyroglobulin plates andthe 15/15 tetanus toxoid and 15/15 thyroglobulin plates are duplicatesfrom each other except for the coating agent. OD values higher thanthree times the background are assured specific. 1 2 3 4 5 6 7 8 9 10 1112 TT-coated plate 10/10 washings A 0.139 0.093 0.089 0.121 0.117 0.5980.146 0.115 0.18 0.155 0.543 0.601 B 0.136 0.404 0.159 0.187 0.489 0.1340.216 0.092 0.222 0.108 0.181 0.484 C 0.197 0.526 0.09 0.213 0.395 0.1550.108 0.12 0.183 0.136 0.092 0.866 D 0.143 0.258 0.101 0.422 0.088 0.2430.485 0.251 0.304 0.198 0.478 0.091 E 0.445 0.169 0.526 0.481 0.2060.285 0.111 0.119 0.128 0.2 0.118 0.098 F 0.237 0.291 0.594 0.139 0.2060.565 0.543 0.091 0.136 0.227 0.228 0.099 G 0.459 0.102 0.152 0.6590.203 0.452 0.152 0.133 0.094 0.102 0.375 0.098 H 0.341 0.623 0.7450.415 0.682 0.527 0.655 0.114 0.258 0.284 0.685 0.113 TT-coated plate15/15 washings A 0.247 0.582 0.421 0.428 0.133 0.082 0.262 0.079 0.3430.414 0.095 0.292 B 0.065 0.364 0.073 0.042 0.049 0.071 0.046 0.1030.078 0.057 0.048 0.155 C 0.081 0.044 0.066 0.082 0.225 0.444 0.2030.362 0.122 0.047 0.052 0.309 D 0.092 0.11 0.59 0.22 0.33 0.544 0.0580.159 0.047 0.174 0.086 0.05  E 0.469 0.577 0.206 0.304 0.13 0.749 0.4310.062 0.167 0.049 0.056 0.049 F 0.846 0.07 0.561 0.656 0.882 0.094 0.3830.13 0.152 0.098 0.134 0.048 G 0.537 0.052 0.49 0.105 0.337 0.193 0.5140.294 0.068 0.35 0.525 0.05  H 0.061 0.306 0.157 0.853 0.054 0.534 0.1020.235 0.441 0.412 0.565 0.061 Thyroglobulin-coated plate 10/10 washingsA 0.047 0.051 0.045 0.043 0.051 0.044 0.046 0.042 0.047 0.048 0.0490.05  B 0.042 0.042 0.042 0.042 0.043 0.041 0.041 0.042 0.043 0.0450.042 0.046 C 0.044 0.043 0.043 0.044 0.043 0.444 0.043 0.042 0.0430.041 0.044 0.046 D 0.045 0.044 0.044 0.044 0.045 0.046 0.045 0.0560.045 0.049 0.048 0.73  E 0.046 0.045 0.046 0.044 0.045 0.044 0.0440.044 0.047 0.046 0.047 0.926 F 0.048 0.045 0.044 0.046 0.044 0.0430.044 0.046 0.046 0.046 0.046 0.792 G 0.051 0.048 0.045 0.045 0.0440.043 0.048 0.045 0.048 0.051 0.045 0.053 H 0.064 0.05 0.049 0.047 0.050.051 0.047 0.046 0.047 0.047 0.047 0.056 Thyroglobulin-coated plate15/15 washings A 0.036 0.049 0.045 0.044 0.046 0.047 0.046 0.042 0.0420.043 0.042 0.041 B 0.045 0.042 0.041 0.043 0.043 0.043 0.045 0.0450.047 0.048 0.044 0.045 C 0.049 0.047 0.047 0.046 0.046 0.046 0.0450.047 0.046 0.045 0.045 0.052 D 0.047 0.049 0.048 0.048 0.048 0.0480.047 0.052 0.048 0.046 0.048 0.456 E 0.049 0.047 0.047 0.047 0.0470.049 0.047 0.048 0.047 0.046 0.048 0.412 F 0.05 0.047 0.046 0.046 0.0460.046 0.046 0.046 0.046 0.047 0.048 0.528 G 0.05 0.048 0.045 0.046 0.0460.049 0.048 0.046 0.053 0.049 0.05 0.057 H 0.057 0.05 0.046 0.047 0.0470.049 0.047 0.047 0.046 0.047 0.053 0.048

TABLE 3 Protein sequence analysis of ELISA positive tetanus toxoidbinders. CDR3 sequence, CDR3 length, VH family members andspecific name, JH origin and DH origin of the clones is indicated. CDR3CDR3/SEQ ID NO: length VH DH JH V Gene family HGAYYTYDEKAWFAY  15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAYYTYDEKAWFAY 15musIGHV192 DSP2.11 JH3 VH7183 (SEQ ID NO: 40) mouse HGAFYTYDEKPWFAY 15musIGHV192 IGHD2- JH3 VH7183 (SEQ ID NO: 41) 14*01 mouse HISYYRYDEEVSFAY15 musIGHV192 IGHD2- JH3 VH7183 (SEQ ID NO: 42) 14*01 mouseHISYYRYDEEVSFAY 15 musIGHV192 IGHD2- JH3 VH7183 (SEQ ID NO: 42) 14*01mouse GWRAFAY 7 musIGHV131 DSP2.9 JH3 VH7183 (SEQ ID NO: 43) mouseGWRAFAY 7 musIGHV131 DSP2.9 JH3 VH7183 (SEQ ID NO: 43) mouse GWRAFAY 7musIGHV131 DSP2.9 JH3 VH7183 (SEQ ID NO: 43) mouse DRGNYYGMDY 10musIGHV178 DSP2.1 JH4 VH7183 (SEQ ID NO: 44) mouse LGDYYVDWFFAV 12musIGHV165 DFL16.1 JH1 VH7183 (SEQ ID NO: 45) mouse NFPAWFAF 8musIGHV547 DST4.3inv JH3 VJH558 (SEQ ID NO: 46) mouse NFPAWFAY 8musIGHV547 DSP2.1 JH3 VJH558 (SEQ ID NO: 46) mouse NFPAWFVY 8 musIGHV547DSP2.1 JH3 VJH558 (SEQ ID NO: 46) mouse SFTPVPFYYGYDWYF 17 musIGHV532DSP2.3 JH1 VJH558 DV mouse (SEQ ID NO: 47) SFTPVPFYYGYDWYF 17 musIGHV532DSP2.3 JH1 VJH558 DV mouse (SEQ ID NO: 47) SDYDWYFDV 9 musIGHV286 DSP2.2JH1 VJH558 (SEQ ID NO: 48) mouse SDYDWYFDV 9 musIGHV286 DSP2.2 JH1VJH558 (SEQ ID NO: 48) mouse DSKWAYYFDY 10 musIGHV532 DST4.3 JH2 VJH558(SEQ ID NO: 49) mouse GDYTGYGMDY 10 musIGHV125 DSP2.13 JH4 VHSM7(SEQ ID NO: 50) mouse GDYTGYGMDY 10 musIGHV125 DSP2.13 JH4 VHSM7(SEQ ID NO: 50) mouse GGYDGYWFPY 10 musIGHV125 DSP2.9 JH3 VHSM7(SEQ ID NO: 51) mouse

TABLE 4 Vector combinations that were transfected to HEK293T. CombinedConc. Code HC vector LC vector vector Prep name (μg/ml) A x0817676_pSELECT_0815426 x PIGKV1-39/ — (IGKV1-39) P1 B x0817678_pSELECT_0815427 x PIGLV2-14/ — (IGLV2-14) P1 C MV11100817676_pSELECT_0815426 x PMV1110/ 11.0 (IGKV1-39) IGKV1-39/P1 D MV11100817678_pSELECT_0815427 x PMV1110/ 15.4 (IGLV2-14) IGLV2-14/P1 E x xMG1494 MG1494/P2 16.1

TABLE 5 HLA allotypes considered in T_(H)-epitope profiling. Thecorresponding serotypes are shown, as well as allotype frequencies inthe Caucasian population (Klitz et al. (2003), Tissue Antigens 62:296-307; Gjertson and Terasake (eds) in: HLA 1997; Gjertson and Terasake(eds) in: HLA 1998; Castelli et al. (2002), J. Immunol. 169: 6928-6934).Frequencies can add up to more than 100% since each individual has twoalleles for each gene. If all allele frequencies of a single gene wereknown, they would add up to slightly less than 200% due to homozygousindividuals. HLA type Serotype Population % HLA type Serotype Population% DRB1*0101 DR1 17.4 DRB4*0103 DR53 21 DRB1*0102 DR1 4.9 DRB5*0101 DR5115.8 DRB1*0301 DR17(3) 21.2 DRB5*0202 DR51 5.7 DRB1*0401 DR4 11.5DQA1*0101/DQB1*0501 DQ5(1) 20.5 DRB1*0402 DR4 3.1 DQA1*0102/DQB1*0502DQ5(1) 2.6 DRB1*0404 DR4 5.5 DQA1*0102/DQB1*0602 DQ6(1) 26.5 DRB1*0405DR4 2.2 DQA1*0102/DQB1*0604 DQ6(1) 6.7 DRB1*0407 DR4 <2DQA1*0103/DQB1*0603 DQ6(1) 11 DRB1*0701 DR7 23.4 DQA1*0104/DQB1*0503DQ5(1) 4 DRB1*0801 DR8 3.3 DQA1*0201/DQB1*0202 DQ2 20.9 DRB1*0802 DR8 <2DQA1*0201/DQB1*0303 DQ9(3) 7.2 DRB1*0901 DR9 <2 DQA1*0301/DQB1*0301DQ7(3) 12.5 DRB1*1101 DR11(5) 17 DQA1*0301/DQB1*0302 DQ8(3) 18.3DRB1*1104 DR11(5) 5.7 DQA1*0401/DQB1*0402 DQ4 4.5 DRB1*1201 DR12(5) 3.1DQA1*0501/DQB1*0201 DQ2 24.6 DRB1*1301 DR13(6) 15.4 DQA1*0501/DQB1*0301DQ7(3) 20.9 DRB1*1302 DR13(6) 10.8 DPA1*0103/DPB1*0201 DPw2 19.9DRB1*1401 DR14(6) 4.2 DPA1*0103/DPB1*0401 DPw4 65.1 DRB1*1501 DR15(2)13.2 DPA1*0103/DPB1*0402 DPw4 24.3 DRB1*1601 DR16(2) 5.5DPA1*0201/DPB1*0101 DPw1 6.3 DRB3*0101 DR52 24.6 DPA1*0201/DPB1*0301DPw3 <2 DRB3*0202 DR52 43 DPA1*0201/DPB1*0501 DPw5 <2 DRB3*0301 DR52 10DPA1*0201/DPB1*0901 — 2.4 DRB4*0101 DR53 25.5

TABLE 6 T_(H) epitope counts for IGKV1-39. Peptides binding to multipleHLAs of the same group (DRB1, DRB3/4/5, DP, DQ) are counted as one.Values between brackets refer to germline filtered peptides DRB1DRB3/4/5 DQ DP Strong Medium Strong Medium Strong Medium Strong MediumMerus IGKV1-39 0 (+6) 0 (+16) 0 (+0) 0 (+5) 0 (+3) 0 (+9) 0 (+0) 0 (+9)

TABLE 7 Mapping of Epibase ® predictions for Mercus IGKV1-39 in theclassical 15-mer peptide format. This table shows the allotype count ofcritical epitopes (SEQ ID NOs: 52-83) and implicated serotypes for eachof the 15-mers spanning the Merus IGKV1-39 sequence. Start 15-merAllotype 15mer position sequence count Implicated serotypes 1 1DIQMTQSPSSLSASV 6 DR1, DR4, DR7, DR9 2 4 MTQSPSSLSASVGDR 5 DR1, DR4, DR93 7 SPSSLSASVGDRVTI 0 4 10 SLSASVGDRVTITCR 0 5 13 ASVGDRVTITCRASQ 0 6 16GDRVTITCRASQSIS 2 DR11(5), DR7 7 19 VTITCRASQSISSYL 4DQ2, DR11(5), DR4, DR7 8 22 TCRASQSISSYLNWY 2 DQ2, DR4 9 25ASQSISSYLNWYQQK 5 DR13(6), DR15(2), DR4 10 28 SISSYLNWYQQKPGK 8DR12(5), DR13(6), DR15(2), DR16(2), DR4, DR8 11 31 SYLNWYQQKPGKAPK 10DR1, DR12(5), DR16(2), DR4, DR51, DR8 12 34 NWYQQKPGKAPKLLI 9DR1, DR15(2),  DR4, DR51, DR8 13 37 QQKPGKAPKLLIYAA 7 DQ4, DR1, DR11(5),DR15(2), DR51, DR8 14 40 PGKAPKLLIYAASSL 7 DQ4, DR1, DR11(5), DR4, DR8 15 43 APKLLIYAASSLQSG 15 DR1, DR11(5), DR12(5), DR13(6),DR14(6), DR15(2), DR4, DR51, DR8, DR9 16 46 LLIYAASSLQSGVPS 15DR1, DR11(5), DR12(5), DR13(6), DR14(6), DR15(2), DR4, DR51, DR8, DR9 1749 YAASLQSGVPSRFS 1 DR15(2) 18 52 SSLQSGVPSRFSGSG 1 DR15(2) 19 55QSGVPSRFSGSGSGT 0 20 58 VPSRFSGSGSGTDFT 0 21 61 RFSGSGSGTDFTLTI 0 22 64GSGSGTDFTLTISSL 1 DR52 23 67 SGTDFTLTISSLQPE 4 DR4, DR52, DR7, DR9 24 70DFTLTISSLQPEDEA 4 DQ2, DR4, DR7, DR9 25 73 LTISSLQPEDFATYY 1 DQ2 26 76SSLQPEDFATYYCQQ 0 27 79 QPEDFATYYCQQSYS 1 DR4 28 82 DFATYYCQQSYSTPP 5DR4, DR51, DR7 29 85 TYYCQQSYSTPPTFG 4 DR4, DR51, DR7 30 88CQQSYSTPPTFGQGT 0 31 91 SYSTPPTFGQGTKVE 0 32 94 TPPTFGQGTKVEIK 0

TABLE 8 The V_(H) gene from PG1433 paired with various light chain geneswith differing rates of amino acid mutation were compared for productionlevels with the original clone containing the IGKV1-39 gene. Number ofLight amino acid concentration IgG name chain gene mutations (μg/ml)PG1433 1-39 0 63, 45.5, 38.6 (avg = 49) PG1631 1-12 4 10.5 PG1632 1-27 79.3 PG1634 1D-12 10 10.8 PG1635 1D-33 6 10.2 PG1642 1-5 8 7.1 PG1644 1-93 7.8 PG1650 1D-39 3 9.1 PG1652 2D-28 3 7.1 PG1653 3-15 14 7 PG1654 3-202 5.2 PG1674 1-40 7 8.2 PG1678 2-11 2 8.1 PG1680 2-14 15 10.8 PG1682 3-113 9.9 PG1683 6-57 6 13.9

TABLE 9 Numbers of lymphocytes harvested from the bone marrow and spleenof wild-type and transgenic mice *10e6/ml total vol total cells cells(ml) *10⁶ Bone Marrow Wt 18.82 5.05 95.0 Wt 19.24 4.96 95.4 CD19-Cre23.42 5.08 119.0 CD19-Cre 20.58 4.82 99.2 CD19-Cre 25.77 5.15 132.7CD19-Cre/HuVk1 17.71 5.06 89.6 CD19-Cre/HuVk1 12.60 5.33 67.2CD19-Cre/HuVk1 18.13 5.27 95.5 Spleen Wt 41.70 5.36 223.5 Wt 37.85 4.71178.3 CD19-Cre 60.19 3.77 226.9 CD19-Cre 35.06 3.66 128.3 CD19-Cre 80.694.60 371.2 CD19-Cre/HuVk1 51.67 4.48 231.5 CD19-Cre/HuVk1 58.80 6.24366.9 CD19-Cre/HuVk1 24.37 6.25 152.3

What is claimed is:
 1. A transgenic mouse whose genome comprises atransgene comprising a human immunoglobulin light chain germlineIGκV1-39 gene segment joined to a human immunoglobulin light chaingermline J gene segment, such that there is no mutation due to saidjoining, wherein the joined IGκV1-39/J gene segments encode a rearrangedhuman immunoglobulin light chain variable region, wherein the transgeneis inserted by site-specific integration in the murine Rosa26-locus orsaid transgene lacks a MoEκi enhancer or comprises a truncated 3′ kappaenhancer or combination of these, wherein said transgene is resistant tosomatic hypermutation, and wherein the transgene comprises a murinelight chain constant region gene segment or is operatively linked to amouse light chain constant region gene segment, wherein the transgenicmouse, in response to an antigen, produces antigen specific antibodiescomprising the rearranged human light chain variable region and a murinelight chain constant region, paired with a diversity of immunoglobulinheavy chains encoded by rearranged and somatically hypermutated heavychain genes, and wherein the endogenous κ light chain locus of thetransgenic mouse is functionally silenced.
 2. The transgenic mouse ofclaim 1, wherein the joined IGκV1-39/J gene segments encode a rearrangedhuman immunoglobulin light chain variable region comprising the aminoacid sequence of the Vκ1-39 region of SEQ ID NO:85.
 3. The transgenicmouse of claim 1, wherein the expression of the rearranged humanimmunoglobulin light chain variable region is under the control of a Bcell specific promoter selected from the group consisting of CD19, CD20,μHC, VpreB1, VpreB2, VpreB3, λ5, Igα, Igβ, κLC, λLC and BSAP (Pax5). 4.The transgenic mouse of claim 1, wherein the transgene comprises, in a5′ to 3′ direction, a promoter selected from the group consisting ofCD19, CD20, μHC, VpreB1, VpreB2, VpreB3, λ5, Igα, Igβ, κLC, λLC and BSAP(Pax5), a human or mouse leader, and a nucleic acid encoding therearranged human immunoglobulin light chain variable region.
 5. Thetransgenic mouse of claim 1, wherein the murine immunoglobulin lightchain constant region is a rat immunoglobulin light chain constantregion.